Compositions and methods for rapid in vitro synthesis of bioconjugate vaccines in vitro via production and N-glycosylation of protein carriers in detoxified prokaryotic cell lysates

ABSTRACT

Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated carrier proteins. The glycosylated carrier proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated carrier proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated carrier proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62/791,425, filed on Jan. 11,2019 and to U.S. Provisional Application No. 62/644,811, filed on Mar.19, 2018, the contents of which are incorporated herein by reference intheir entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under MCB1413563 awardedby the National Science Foundation and HDTRA1-15-1-0052 awarded by theDepartment of Defense. The government has certain rights in theinvention.

SEQUENCE LISTING

A Sequence Listing accompanies this application and is submitted as anASCII text file of the sequence listing named “702581_00836_ST25.txt”which is 35,544 bytes in size and was created on Apr. 20, 2022. Thesequence listing is electronically submitted via EFS-Web with theapplication and is incorporated herein by reference in its entirety.

BACKGROUND

The field of the invention relates to in vitro synthesis ofN-glycosylated protein in prokaryotic cell lysates. In particular, thefield of the invention relates to the use of N-glycosylated proteinssynthesized in vitro in prokaryotic cell lysates as vaccine conjugatesagainst pathogens such as bacteria.

Conjugate vaccines are among the safest and most effective methods forprevention of life-threatening bacterial infections. Bioconjugatevaccines are a type of conjugate vaccine produced via protein glycancoupling technology (PGCT), in which polysaccharide antigens areconjugated via N-glycosylation to recombinant carrier proteins using abacterial oligosaccharyltransferase (OST) in living Escherichia colicells. Bioconjugate vaccines have the potential to greatly reduce thetime and cost required to produce antibacterial vaccines. However, PGCTis limited by: i) the length of in vivo process development timelines;and ii) the fact that FDA-approved carrier proteins, such as the toxinsfrom Clostridium tetani and Corynebacterium diptheriae, have not yetbeen demonstrated to be compatible with N-linked glycosylation in livingE. coli. Here, we have applied cell-free glycoprotein synthesis (CFGpS)technology to enable rapid in vitro production of bioconjugate vaccinesagainst pathogenic strains of Escherichia coli and Franscicellatularensis in reactions lasting 20 hours. Due to the modular nature ofthe CFGpS system, this cell-free strategy could be easily applied toproduce bioconjugates using FDA-approved carrier proteins or additionalvaccines against pathogenic bacteria whose surface antigen gene clustersare known. We further show that this system can be lyophilized andretain bioconjugate synthesis capability, demonstrating the potentialfor on-demand vaccine production and development in resource-poorsettings. This work represents the first demonstration of bioconjugatevaccine production in E. coli lysates and has promising applications asa portable prototyping or production platform for antibacterial vaccinecandidates.

SUMMARY

Disclosed are methods, systems, components, and compositions forcell-free synthesis of glycosylated carrier proteins. The glycosylatedcarrier proteins may be utilized in vaccines, including anti-bacterialvaccines.

The glycosylated carrier proteins may include a bacterial polysaccharideconjugated to a carrier, which may be utilized to generate an immuneresponse in an immunized host against the polysaccharide conjugated tothe carrier. Suitable carriers may include but are not limited toHaemophilus influenzae protein D (PD), Neisseria meningitidis porinprotein (PorA), Corynebacterium diphtherias toxin (CRM197), Clostridiumtetani toxin (TT), and Escherichia coli maltose binding protein, orvariants thereof.

The glycosylated carrier proteins may be synthesized in cell-freeglycoprotein synthesis (CFGpS) systems using detoxified prokaryote celllysates that are enriched in components for glycoprotein synthesis suchas oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides(LLOs) including OSTs and. LLOs associated with synthesis of bacterialO-antigens. As such, the prokaryote cell lysates may be prepared fromrecombinant prokaryotes that have been engineered to express detoxifiedlipid A, and recombinant prokaryotes that have been engineered toexpress heterologous OSTs and/or that have been engineered to expressheterologous glycan synthesis pathways for production of LLOs. Thedisclosed lysates may be described as modular and may be combined toprepare glycosylated proteins in the disclosed CFGpS systems.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . WAX platform enables on-demand and portable production ofantibacterial vaccines. The in vitro bioconjugate vaccine expression(iVAX) platform provides a rapid means to develop and distributeantibacterial vaccines in the event of a pathogen outbreak. Expressionof pathogen-specific polysaccharides (e.g., CPS, LPS) and a bacterialoligosaccharyltransferases enzyme in engineered strains with detoxifiedlipid A yields low-endotoxin lysates containing all of the machineryrequired for synthesis of bioconjugate vaccines. Reactions catalyzed byiVAX lysates can be used to produce bioconjugates containingFDA-approved carrier proteins and can be freeze-dried without loss ofactivity for refrigeration-free transportation and storage. Freeze-driedreactions can be activated at the point of need via simple rehydrationand reproducibly synthesize immunologically active bioconjugates in ˜1h.

FIG. 2 . In vitro synthesis of FDA-approved carrier proteins. (a) Allfour FDA-approved conjugate vaccine carrier proteins were synthesizedsolubly in vitro, as measured via ¹⁴C-leucine incorporation. Theseinclude H. influenzae protein D (PD), the N. meningitidis porin protein(PorA), and genetically detoxified variants of the C. diphtherias toxin(CRM197) and the C. tetani toxin (TT). Promising carriers that are notyet FDA-approved were also synthesized solubly, including E. colimaltose binding protein (MBP) and the fragment C (TTc) and light chain(TTlight) domains of TT. Values represent means and error bars representstandard deviations of biological replicates (n=3). (b) Full lengthproduct was observed for all proteins tested via Western blot. Differentexposures are indicated with solid lines.

FIG. 3 . On-demand and reproducible production of bioconjugates againstF. tularensis using iVAX. iVAX lysates were prepared from cellsexpressing CjPg1B and a biosynthetic pathway encoding FtO-PS. (a)Glycosylation of sfGFP^(217-DQNAT) with FtO-PS was only observed whenCjPg1B, FtO-PS, and the preferred sequon were present in the reaction(lane 3). When plasmid DNA is omitted, sfGFP^(217-DQNAT) synthesis wasnot observed. (b) Biological replicates of iVAX reactions producingsfGFP^(217-DQNAT) using the same lot (left) or different lots (right) ofiVAX lysates demonstrated reproducibility of reactions and lysatepreparation. (c) On-demand synthesis of bioconjugate vaccines withimmunogenic carriers including authentic FDA-approved carriers.Bioconjugates were purified using Ni-NTA agarose from 1 mL iVAXreactions lasting ˜1 h at 30° C. Unless replicates are explicitly shown,images are representative of at least three biological replicates.Dashed lines indicate samples are from the same blot with the sameexposure.

FIG. 4 . Detoxified, lyophilized iVAX reactions produce bioconjugatesthat are comparable to those made using state-of-the-art technologies.(a) iVAX lysates were detoxified via deletion of lpxM and expression ofF. tularensis LpxE in the source strain for lysate production. Thisstrain engineering resulted in production of a pentaacylated,monophosporylated lipid A molecule with reduced endotoxin activity.*p=0.019 and **p=0.003, as determined by two-tailed t-test. (b)Identical iVAX reactions producing sfGFP^(217-DQNAT) were runimmediately or following lyophilization and rehydration. Glycosylationactivity was preserved following lyophilization, demonstrating thepotential of iVAX reactions for portable biosynthesis of bioconjugatevaccines.

FIG. 5 . iVAX-derived bioconjugates elicit bactericidal antibodiesspecific to FtLPS in mice. (a) FtLPS-specific IgG titers were measuredby ELISA in endpoint (day 70) serum of individual mice (black dots) andmean titers of each group (red lines). Six groups of BALB/c mice wereimmunized subcutaneously with PBS or 7.5 μg of purified, cell-freesynthesized aglycosylated MBP^(4×DQNAT) FtO-PS-conjugated MBP^(4×DQNAT),aglycosylated PD^(4×DQNAT), or FtO-PS-conjugated PD^(4×DQNAT).FtO-PS-conjugated MBP^(4×DQNAT) prepared in living E. coli cells usingPCGT was used as a positive control. Each group was composed of six miceexcept for the PBS control group, which was composed of five mice. Micewere boosted on days 21 and 42 with identical doses of antigen.iVAX-derived bioconjugates elicited significantly higher levels ofFtLPS-specific IgG compared to all other groups (**p<0.01, Tukey-KramerHSD). (b) IgG1 and IgG2a subtype titers measured by ELISA from endpointserum revealed that iVAX-derived bioconjugates boosted production ofFtO-PS-specific IgG1 compared to all other groups tested (**p<0.01,Tukey-Kramer HSD). This indicates that iVAX bioconjugates elicited aTh2-biased immune response typical of most conjugate vaccines. Valuesrepresent means and error bars represent standard errors ofFtLPS-specific IgGs detected by ELISA. (c) FtLPS-specific IgGs arebactericidal against the F. tularensis LVS. Values represent averagesand error bars represent standard deviations of bactericidal activityfrom individual mice in each immunization group.

FIG. 6 . Protein synthesis and glycosylation in iVAX reactions occurs in1 h and is robust at a range of temperatures. (a) With the exception ofCRM197, all carriers were expressed with similar soluble yields at 25°C., 30° C., and 37° C., as measured via ¹⁴C-leucine incorporation.Kinetics of O-PS glycosylation in iVAX reactions at (b) 30° C. and (c)37° C., 25° C., and room temperature (˜21° C.) are comparable and showedthat protein synthesis and glycosylation occurred in the first hour ofthe iVAX reaction. Together, these results demonstrate that stricttemperature control is not required for bioconjugate vaccine synthesisin the iVAX platform, which is critical for a decentralized productionplatform in settings where precise temperature control is not alwaysfeasible.

FIG. 7 . Optimization of PorA and tetanus toxin expression in vitro.Blots show soluble fractions of reactions producing PorA^(4×DQNAT) orTT^(4×DQNAT). (a) Soluble expression of PorA was improved through theaddition of lipid nanodiscs to the reaction. (b) Expression offull-length TT was enhanced by (i) performing in vitro protein synthesisin oxidizing conditions to improve assembly of the disulfide-bondedheavy and light chains into full-length TT and (ii) allowing reactionsto run for only 2 h to minimize protease degradation. Images arerepresentative of at least three biological replicates. Dashed lineindicates samples are from the same blot with the same exposure.

FIG. 8 . In vitro synthesis of CRM197 and TT compared to commercialstandards. In vitro synthesized (a) CRM197 and (b) TT are detected withα-DT and α-TT antibodies, respectively, and are comparable in size tocommercially available purified DT and TT protein standards (50 ngstandard loaded). Images are representative of at least three biologicalreplicates. Dashed lines indicate samples are from the same blot withthe same exposure. Solid lines indicate different exposures.

FIG. 9 . Detailed Western blot information for FIG. 3 a . The anti-Hisand anti-FtO-PS signals are shown separately as a representativeglycosylation Western blot. Images are representative of at least threebiological replicates.

FIG. 10 . Production of bioconjugates against F. tularensis using PGCTin living E. coli. Bioconjugates were produced via PGCT in CLM24 cellsexpressing CjPg1B, the biosynthetic pathway for FtO-PS, and a panel ofimmunogenic carriers including authentic FDA-approved carriers. Weobserved low expression of PorA, a membrane protein, and limitedglycosylation and laddering of conjugated FtO-PS in all carriers usingthis method. Images are representative of at least three biologicalreplicates.

FIG. 11 . WAX reactions produce clinically relevant amounts ofbioconjugates in 1 h. Protein synthesis and modification with FtO-PSwere measured in reactions producing MBP^(4×DQNAT) and PD^(4×DQNAT).After ˜1 h, reactions produced ˜40 μg mL⁻¹ protein, as measured via¹⁴C-leucine incorporation, of which ˜20 μg mL⁻¹ was modified with theFtO-PS, as determined by densitometry. Values represent means and errorbars represent standard errors of biological replicates (n=2).

FIG. 12 . The iVAX platform is modular and can be used to synthesizediverse bioconjugates. iVAX lysates were prepared from cells expressingCjPg1B and biosynthetic pathways for either (a) the E. coli O78 antigenor (b) the E. coli O7 antigen and used to synthesize PD^(4×DQNAT) (top)or sfGFP^(217-DQNAT) (bottom) bioconjugates. The structure andcomposition of the repeating monomer unit for each antigen is shown.Both polysaccharide antigens are compositionally and, in the case of theO7 antigen, structurally distinct compared to the F. tularensis Oantigen. If a commercial anti-O-PS serum or antibody was available, itwas used to confirm the identity of the conjugated O antigen (α-EcO78blots, panel a). Asterisk denotes bands resulting from non-specificserum antibody binding. Images are representative of at least threebiological replicates. Dashed lines indicate samples are from the sameblot with the same exposure.

FIG. 13 . Lipid A remodeling does not affect glycosylation activity iniVAX reactions. iVAX lysates were prepared from either wild-type CLM24,CLM24 ΔlpxM, or CLM24 ΔlpxM expressing FtLpxE cells also expressingCjPg1B and FtO-PS. These all-in-one lysates synthesizing versions of thelipid A molecule with varying structures and toxicities (FIG. 4 a ) wereused in iVAX reactions primed with plasmid encoding sfGFP^(217-DQNAT).No significant difference in glycosylation activity was observed. Imagesare representative of at least three biological replicates.

FIG. 14 . Scaling of detoxified iVAX lysate production and freeze-driedreactions is reproducible. (a) To generate material for immunizations,fermentations to produce detoxified iVAX lysates were scaled from 0.5 Lto 10 L. iVAX reactions were prepared using lysates produced at small orlarge scale and primed with plasmid encoding sfGFP^(217-DQNAT). Weobserved similar levels of glycosylation from lysates derived from 0.5 Land 10 L cultures, and across different batches of lysate produced from10 L fermentations. (b) For immunizations, we prepared two lots ofFtO-PS-conjugated MBP^(4×DQNAT) and PD^(4×DQNAT) from 5 mL freeze-driediVAX reactions. We observed similar levels of purified protein (˜200 μg)and FtO-PS modification (>50%, measured by densitometry) across bothcarriers and lots of material.

FIG. 15 . FtLPS-specific antibody titers in vaccinated mice over time.Six groups of BALB/c mice were immunized subcutaneously with PBS or 7.5μg of purified, cell-free synthesized aglycosylated MBP^(4×DQNAT),FtO-PS-conjugated MBP^(4×DQNAT), aglycosylated PD^(4×DQNAT), orFtO-PS-conjugated PD^(4×DQNAT). FtO-PS-conjugated MBP^(4×DQNAT) preparedin living E. coli cells using PCGT was used as a positive control. Eachgroup was composed of six mice except for the PBS control group, whichwas composed of five mice. Mice were boosted on days 21 and 42 withidentical doses of antigen. FtLPS-specific IgG titers were measured byELISA in serum collected on day −1, 35, 49, 63, and 70 following initialimmunization. iVAX-derived bioconjugates elicited significantly higherlevels of FtLPS-specific IgG compared to compared to the PBS controlgroup in serum collected on day 35, 49, and 70 of the study (**p<0.01,Tukey-Kramer HSD). Values represent means and error bars representstandard errors of FtLPS-specific IgGs detected by ELISA.

FIG. 16 . DNA concentration in iVAX reactions can be reduced withoutimpacting protein synthesis yields or kinetics. iVAX reactions wereprepared containing 13.33, 6.67, 3.33, or 1.33 ng/μL plasmid DNAtemplate encoding sfGFP. We observed that both (a) protein synthesisyields after 20 h and (b) initial rates of protein synthesis wereconserved with 13.33 or 6.67 ng/μL DNA template. At lower DNAconcentrations, DNA template appeared to be limiting as lower proteinsynthesis yields and initial rates were observed.

DETAILED DESCRIPTION

The present invention is described herein using several definitions, asset forth below and throughout the application.

Definitions

The disclosed subject matter may be further described using definitionsand terminology as follows. The definitions and terminology used hereinare for the purpose of describing particular embodiments only, and arenot intended to be limiting.

As used in this specification and the claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise. For example, the term “a gene” or “an oligosaccharide” shouldbe interpreted to mean “one or more genes” and “one or moreoligosaccharides,” respectively, unless the context clearly dictatesotherwise. As used herein, the term “plurality” means “two or more.”

As used herein, “about”, “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” and“approximately” will mean up to plus or minus 10% of the particular termand “substantially” and “significantly” will mean more than plus orminus 10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.” The terms “comprise”and “comprising” should be interpreted as being “open” transitionalterms that permit the inclusion of additional components further tothose components recited in the claims. The terms “consist” and“consisting of” should be interpreted as being “closed” transitionalterms that do not permit the inclusion of additional components otherthan the components recited in the claims. The term “consistingessentially of” should be interpreted to be partially closed andallowing the inclusion only of additional components that do notfundamentally alter the nature of the claimed subject matter.

The phrase “such as” should be interpreted as “for example, including.”Moreover the use of any and all exemplary language, including but notlimited to “such as”, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed.

Furthermore, in those instances where a convention analogous to “atleast one of A, B and C, etc.” is used, in general such a constructionis intended in the sense of one having ordinary skill in the art wouldunderstand the convention (e.g., “a system having at least one of A, Band C” would include but not be limited to systems that have A alone, Balone, C alone, A and B together, A and C together, B and C together,and/or A, B, and C together.). It will be further understood by thosewithin the art that virtually any disjunctive word and/or phrasepresenting two or more alternative terms, whether in the description orfigures, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or ‘B or “A and B.”

All language such as “up to,” “at least,” “greater than,” “less than,”and the like, include the number recited and refer to ranges which cansubsequently be broken down into ranges and subranges. A range includeseach individual member. Thus, for example, a group having 1-3 membersrefers to groups having 1, 2, or 3 members. Similarly, a group having 6members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

The modal verb “may” refers to the preferred use or selection of one ormore options or choices among the several described embodiments orfeatures contained within the same. Where no options or choices aredisclosed regarding a particular embodiment or feature contained in thesame, the modal verb “may” refers to an affirmative act regarding how tomake or use and aspect of a described embodiment or feature contained inthe same, or a definitive decision to use a specific skill regarding adescribed embodiment or feature contained in the same. In this lattercontext, the modal verb “may” has the same meaning and connotation asthe auxiliary verb “can.”

As used herein, the terms “bind,” “binding,” “interact,” “interacting,”“occupy” and “occupying” refer to covalent interactions, noncovalentinteractions and steric interactions. A covalent interaction is achemical linkage between two atoms or radicals formed by the sharing ofa pair of electrons (a single bond), two pairs of electrons (a doublebond) or three pairs of electrons (a triple bond). Covalent interactionsare also known in the art as electron pair interactions or electron pairbonds. Noncovalent interactions include, but are not limited to, van derWaals interactions, hydrogen bonds, weak chemical bonds (via short-rangenoncovalent forces), hydrophobic interactions, ionic bonds and the like.A review of noncovalent interactions can be found in Alberts et al., inMolecular Biology of the Cell, 3d edition, Garland Publishing, 1994.Steric interactions are generally understood to include those where thestructure of the compound is such that it is capable of occupying a siteby virtue of its three dimensional structure, as opposed to anyattractive forces between the compound and the site.

Polynucleotides and Synthesis Methods

The terms “nucleic acid” and “oligonucleotide,” as used herein, refer topolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and to any other type ofpolynucleotide that is an N glycoside of a purine or pyrimidine base.There is no intended distinction in length between the terms “nucleicacid”, “oligonucleotide” and “polynucleotide”, and these terms will beused interchangeably. These terms refer only to the primary structure ofthe molecule. Thus, these terms include double- and single-stranded DNA,as well as double- and single-stranded RNA. For use in the presentmethods, an oligonucleotide also can comprise nucleotide analogs inwhich the base, sugar, or phosphate backbone is modified as well asnon-purine or non-pyrimidine nucleotide analogs.

Oligonucleotides can be prepared by any suitable method, includingdirect chemical synthesis by a method such as the phosphotriester methodof Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiestermethod of Brown et al., 1979, Meth. Enzymol. 68:109-151; thediethylphosphoramidite method of Beaucage et al., 1981, TetrahedronLetters 22:1859-1862; and the solid support method of U.S. Pat. No.4,458,066, each incorporated herein by reference. A review of synthesismethods of conjugates of oligonucleotides and modified nucleotides isprovided in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187,incorporated herein by reference.

The term “amplification reaction” refers to any chemical reaction,including an enzymatic reaction, which results in increased copies of atemplate nucleic acid sequence or results in transcription of a templatenucleic acid Amplification reactions include reverse transcription, thepolymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat.Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)), and the ligase chain reaction(LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary“amplification reactions conditions” or “amplification conditions”typically comprise either two or three step cycles. Two-step cycles havea high temperature denaturation step followed by ahybridization/elongation (or ligation) step. Three step cycles comprisea denaturation step followed by a hybridization step followed by aseparate elongation step.

The terms “target,” “target sequence”, “target region”, and “targetnucleic acid,” as used herein, are synonymous and refer to a region orsequence of a nucleic acid which is to be amplified, sequenced, ordetected.

The term “hybridization,” as used herein, refers to the formation of aduplex structure by two single-stranded nucleic acids due tocomplementary base pairing. Hybridization can occur between fullycomplementary nucleic acid strands or between “substantiallycomplementary” nucleic acid strands that contain minor regions ofmismatch. Conditions under which hybridization of fully complementarynucleic acid strands is strongly preferred are referred to as “stringenthybridization conditions” or “sequence-specific hybridizationconditions”. Stable duplexes of substantially complementary sequencescan be achieved under less stringent hybridization conditions;

the degree of mismatch tolerated can be controlled by suitableadjustment of the hybridization conditions. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length andbase pair composition of the oligonucleotides, ionic strength, andincidence of mismatched base pairs, following the guidance provided bythe art (see, e.g., Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol.26(3/4):227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353,which are incorporated herein by reference).

The term “primer,” as used herein, refers to an oligonucleotide capableof acting as a point of initiation of DNA synthesis under suitableconditions. Such conditions include those in which synthesis of a primerextension product complementary to a nucleic acid strand is induced inthe presence of four different nucleoside triphosphates and an agent forextension (for example, a DNA polymerase or reverse transcriptase) in anappropriate buffer and at a suitable temperature.

A primer is preferably a single-stranded DNA. The appropriate length ofa primer depends on the intended use of the primer but typically rangesfrom about 6 to about 225 nucleotides, including intermediate ranges,such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25to 150 nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatenucleic acid, but must be sufficiently complementary to hybridize withthe template. The design of suitable primers for the amplification of agiven target sequence is well known in the art and described in theliterature cited herein.

Primers can incorporate additional features which allow for thedetection or immobilization of the primer but do not alter the basicproperty of the primer, that of acting as a point of initiation of DNAsynthesis. For example, primers may contain an additional nucleic acidsequence at the 5′ end which does not hybridize to the target nucleicacid, but which facilitates cloning or detection of the amplifiedproduct, or which enables transcription of RNA (for example, byinclusion of a promoter) or translation of protein (for example, byinclusion of a 5′-UTR, such as an Internal Ribosome Entry Site (IRES) ora 3′-UTR element, such as a poly(A)_(n) sequence, where n is in therange from about 20 to about 200). The region of the primer that issufficiently complementary to the template to hybridize is referred toherein as the hybridizing region.

As used herein, a primer is “specific,” for a target sequence if, whenused in an amplification reaction under sufficiently stringentconditions, the primer hybridizes primarily to the target nucleic acid.Typically, a primer is specific for a target sequence if theprimer-target duplex stability is greater than the stability of a duplexformed between the primer and any other sequence found in the sample.One of skill in the art will recognize that various factors, such assalt conditions as well as base composition of the primer and thelocation of the mismatches, will affect the specificity of the primer,and that routine experimental confirmation of the primer specificitywill be needed in many cases. Hybridization conditions can be chosenunder which the primer can form stable duplexes only with a targetsequence. Thus, the use of target-specific primers under suitablystringent amplification conditions enables the selective amplificationof those target sequences that contain the target primer binding sites.

As used herein, a “polymerase” refers to an enzyme that catalyzes thepolymerization of nucleotides. “DNA polymerase” catalyzes thepolymerization of deoxyribonucleotides. Known DNA polymerases include,for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNApolymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNApolymerase, among others. “RNA polymerase” catalyzes the polymerizationof ribonucleotides. The foregoing examples of DNA polymerases are alsoknown as DNA-dependent DNA polymerases. RNA-dependent DNA polymerasesalso fall within the scope of DNA polymerases. Reverse transcriptase,which includes viral polymerases encoded by retroviruses, is an exampleof an RNA-dependent DNA polymerase. Known examples of RNA polymerase(“RNAP”) include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6RNA polymerase and E. coli RNA polymerase, among others. The foregoingexamples of RNA polymerases are also known as DNA-dependent RNApolymerase. The polymerase activity of any of the above enzymes can bedetermined by means well known in the art.

The term “promoter” refers to a cis-acting DNA sequence that directs RNApolymerase and other trans-acting transcription factors to initiate RNAtranscription from the DNA template that includes the cis-acting DNAsequence.

As used herein, the term “sequence defined biopolymer” refers to abiopolymer having a specific primary sequence. A sequence definedbiopolymer can be equivalent to a genetically-encoded defined biopolymerin cases where a gene encodes the biopolymer having a specific primarysequence.

As used herein, “expression template” and/or “transcription template”refers to a nucleic acid that serves as substrate for transcribing atleast one RNA that can be translated into a sequence defined biopolymer(e.g., a polypeptide or protein). Expression templates include nucleicacids composed of DNA or RNA. Suitable sources of DNA for use a nucleicacid for an expression template include genomic DNA, cDNA and RNA thatcan be converted into cDNA. Genomic DNA, cDNA and RNA can be from anybiological source, such as a tissue sample, a biopsy, a swab, sputum, ablood sample, a fecal sample, a urine sample, a scraping, among others.The genomic DNA, cDNA and RNA can be from host cell or virus origins andfrom any species, including extant and extinct organisms. As usedherein, “expression template” and “transcription template” have the samemeaning and are used interchangeably.

In certain exemplary embodiments, vectors such as, for example,expression vectors, containing a nucleic acid encoding one or more rRNAsor reporter polypeptides and/or proteins described herein are provided.As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid,” which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Such vectors are referred to herein as “expression vectors.” Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids. In the present specification, “plasmid”and “vector” can be used interchangeably. However, the disclosed methodsand compositions are intended to include such other forms of expressionvectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

In certain exemplary embodiments, the recombinant expression vectorscomprise a nucleic acid sequence (e.g., a nucleic acid sequence encodingone or more rRNAs or reporter polypeptides and/or proteins describedherein) in a form suitable for expression of the nucleic acid sequencein one or more of the methods described herein, which means that therecombinant expression vectors include one or more regulatory sequenceswhich is operatively linked to the nucleic acid sequence to beexpressed. Within a recombinant expression vector, “operably linked” isintended to mean that the nucleotide sequence encoding one or more rRNAsor reporter polypeptides and/or proteins described herein is linked tothe regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription and/ortranslation system). The term “regulatory sequence” is intended toinclude promoters, enhancers and other expression control elements(e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990).

Oligonucleotides and polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides. Examples of modified nucleotides include, but are notlimited to diaminopurine, S²T, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,2,6-diaminopurine and the like. Nucleic acid molecules may also bemodified at the base moiety (e.g., at one or more atoms that typicallyare available to form a hydrogen bond with a complementary nucleotideand/or at one or more atoms that are not typically capable of forming ahydrogen bond with a complementary nucleotide), sugar moiety orphosphate backbone.

As utilized herein, a “deletion” means the removal of one or morenucleotides relative to the native polynucleotide sequence. Theengineered strains that are disclosed herein may include a deletion inone or more genes (e.g., a deletion in gmd and/or a deletion in waaL).Preferably, a deletion results in a non-functional gene product. Asutilized herein, an “insertion” means the addition of one or morenucleotides to the native polynucleotide sequence. The engineeredstrains that are disclosed herein may include an insertion in one ormore genes (e.g., an insertion in gmd and/or an insertion in waaL).Preferably, a deletion results in a non-functional gene product. Asutilized herein, a “substitution” means replacement of a nucleotide of anative polynucleotide sequence with a nucleotide that is not native tothe polynucleotide sequence. The engineered strains that are disclosedherein may include a substitution in one or more genes (e.g., asubstitution in gmd and/or a substitution in waaL). Preferably, asubstitution results in a non-functional gene product, for example,where the substitution introduces a premature stop codon (e.g., TAA,TAG, or TGA) in the coding sequence of the gene product. In someembodiments, the engineered strains that are disclosed herein mayinclude two or more substitutions where the substitutions introducemultiple premature stop codons (e.g., TAATAA, TAGTAG, or TGATGA).

In some embodiments, the engineered strains disclosed herein may beengineered to include and express one or heterologous genes. As would beunderstood in the art, a heterologous gene is a gene that is notnaturally present in the engineered strain as the strain occurs innature. A gene that is heterologous to E. coli is a gene that does notoccur in E. coli and may be a gene that occurs naturally in anothermicroorganism (e.g. a gene from C. jejuni) or a gene that does not occurnaturally in any other known microorganism (i.e., an artificial gene).

Peptides, Polypeptides, Proteins, and Synthesis Methods

As used herein, the terms “peptide,” “polypeptide,” and “protein,” referto molecules comprising a chain a polymer of amino acid residues joinedby amide linkages. The term “amino acid residue,” includes but is notlimited to amino acid residues contained in the group consisting ofalanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D),glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G),histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine(Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Proor P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S),threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), andtyrosine (Tyr or Y) residues. The term “amino acid residue” also mayinclude nonstandard or unnatural amino acids. The term “amino acidresidue” may include alpha-, beta-, gamma-, and delta-amino acids.

In some embodiments, the term “amino acid residue” may includenonstandard or unnatural amino acid residues contained in the groupconsisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine,3-Aminoadipic acid, Hydroxylysine, β-alanine, β-Amino-propionic acid,allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline,4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproicacid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine,2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyricacid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine,2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline,2,2′-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid,Ornithine, and N-Ethylglycine. The term “amino acid residue” may includeL isomers or D isomers of any of the aforementioned amino acids.

Other examples of nonstandard or unnatural amino acids include, but arenot limited, to a p-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, anO-methyl-L-tyrosine, a p-propargyloxyphenylalanine, ap-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine,a tri-O-acetyl-G1cNAcpβ-serine, an L-Dopa, a fluorinated phenylalanine,an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, ap-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnaturalanalogue of a tyrosine amino acid; an unnatural analogue of a glutamineamino acid; an unnatural analogue of a phenylalanine amino acid; anunnatural analogue of a serine amino acid; an unnatural analogue of athreonine amino acid; an unnatural analogue of a methionine amino acid;an unnatural analogue of a leucine amino acid; an unnatural analogue ofa isoleucine amino acid; an alkyl, aryl, acyl, azido, cyano, halo,hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl,seleno, ester, thioacid, borate, boronate, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or aminosubstituted amino acid, or a combination thereof; an amino acid with aphotoactivatable cross-linker; a spin-labeled amino acid; a fluorescentamino acid; a metal binding amino acid; a metal-containing amino acid; aradioactive amino acid; a photocaged and/or photoisomerizable aminoacid; a biotin or biotin-analogue containing amino acid; a ketocontaining amino acid; an amino acid comprising polyethylene glycol orpolyether; a heavy atom substituted amino acid; a chemically cleavableor photocleavable amino acid; an amino acid with an elongated sidechain; an amino acid containing a toxic group; a sugar substituted aminoacid; a carbon-linked sugar-containing amino acid; a redox-active aminoacid; an α-hydroxy containing acid; an amino thio acid; an α,αdisubstituted amino acid; a β-amino acid; a γ-amino acid, a cyclic aminoacid other than proline or histidine, and an aromatic amino acid otherthan phenylalanine, tyrosine or tryptophan.

As used herein, a “peptide” is defined as a short polymer of aminoacids, of a length typically of 20 or less amino acids, and moretypically of a length of 12 or less amino acids (Garrett & Grisham,Biochemistry, 2^(nd) edition, 1999, Brooks/Cole, 110). In someembodiments, a peptide as contemplated herein may include no more thanabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 amino acids. A polypeptide, also referred to as a protein, istypically of length≥100 amino acids (Garrett & Grisham, Biochemistry,2^(nd) edition, 1999, Brooks/Cole, 110). A polypeptide, as contemplatedherein, may comprise, but is not limited to, 100, 101, 102, 103, 104,105, about 110, about 120, about 130, about 140, about 150, about 160,about 170, about 180, about 190, about 200, about 210, about 220, about230, about 240, about 250, about 275, about 300, about 325, about 350,about 375, about 400, about 425, about 450, about 475, about 500, about525, about 550, about 575, about 600, about 625, about 650, about 675,about 700, about 725, about 750, about 775, about 800, about 825, about850, about 875, about 900, about 925, about 950, about 975, about 1000,about 1100, about 1200, about 1300, about 1400, about 1500, about 1750,about 2000, about 2250, about 2500 or more amino acid residues.

A peptide as contemplated herein may be further modified to includenon-amino acid moieties. Modifications may include but are not limitedto acylation (e.g., O-acylation (esters), N-acylation (amides),S-acylation (thioesters)), acetylation (e.g., the addition of an acetylgroup, either at the N-terminus of the protein or at lysine residues),formylation lipoylation (e.g., attachment of a lipoate, a C8 functionalgroup), myristoylation (e.g., attachment of myristate, a C14 saturatedacid), palmitoylation (e.g., attachment of palmitate, a C16 saturatedacid), alkylation (e.g., the addition of an alkyl group, such as anmethyl at a lysine or arginine residue), isoprenylation or prenylation(e.g., the addition of an isoprenoid group such as farnesol orgeranylgeraniol), amidation at C-terminus, glycosylation (e.g., theaddition of a glycosyl group to either asparagine, hydroxylysine,serine, or threonine, resulting in a glycoprotein). Distinct fromglycation, which is regarded as a nonenzymatic attachment of sugars,polysialylation (e.g., the addition of polysialic acid), glypiation(e.g., glycosylphosphatidylinositol (GPI) anchor formation,hydroxylation, iodination (e.g., of thyroid hormones), andphosphorylation (e.g., the addition of a phosphate group, usually toserine, tyrosine, threonine or histidine). Modifications may include theaddition of a glycosylation tag (e.g., 4×DQNAT (SEQ ID NO: 12)optionally which may be added to the N-terminus, C-terminus, or bothtermini) and/or a histidine tag (e.g., 6×His optionally which may beadded to the N-terminus, C-terminus, or both termini).

Reference may be made herein to peptides, polypeptides, proteins andvariants thereof. Reference amino acid sequences may include, but arenot limited to, the amino acid sequence of any of SEQ ID NOs:1-10.Variants as contemplated herein may have an amino acid sequence thatincludes conservative amino acid substitutions relative to a referenceamino acid sequence. For example, a variant peptide, polypeptide, orprotein as contemplated herein may include conservative amino acidsubstitutions and/or non-conservative amino acid substitutions relativeto a reference peptide, polypeptide, or protein. “Conservative aminoacid substitutions” are those substitutions that are predicted tointerfere least with the properties of the reference peptide,polypeptide, or protein, and “non-conservative amino acid substitution”are those substitution that are predicted to interfere most with theproperties of the reference peptide, polypeptide, or protein. In otherwords, conservative amino acid substitutions substantially conserve thestructure and the function of the reference peptide, polypeptide, orprotein. The following table provides a list of exemplary conservativeamino acid substitutions.

TABLE 1 Original Residue Conservative Substitution Ala Gly, Ser Arg His,Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His GluAsp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, ValLys Arg, Gln, Glu Met Leu, Ile Phe Mis, Met, Leu, Trp, Tyr Ser Cys, ThrThr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

Conservative amino acid substitutions generally maintain: (a) thestructure of the peptide, polypeptide, or protein backbone in the areaof the substitution, for example, as a beta sheet or alpha helicalconformation, (b) the charge or hydrophobicity of the molecule at thesite of the substitution, and/or (c) the bulk of the side chain.Non-conservative amino acid substitutions generally disrupt: (a) thestructure of the peptide, polypeptide, or protein backbone in the areaof the substitution, for example, as a beta sheet or alpha helicalconformation, (b) the charge or hydrophobicity of the molecule at thesite of the substitution, and/or (c) the bulk of the side chain.

Variants comprising deletions relative to a reference amino acidsequence of peptide, polypeptide, or protein are contemplated herein. A“deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides relative to a reference sequence. A deletion removes atleast 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues ornucleotides. A deletion may include an internal deletion or a terminaldeletion (e.g., an N-terminal truncation or a C-terminal truncation of areference polypeptide or a 5′-terminal or 3′-terminal truncation of areference polynucleotide).

Variants comprising fragment of a reference amino acid sequence of apeptide, polypeptide, or protein are contemplated herein. A “fragment”is a portion of an amino acid sequence which is identical in sequence tobut shorter in length than a reference sequence. A fragment may compriseup to the entire length of the reference sequence, minus at least oneamino acid residue. For example, a fragment may comprise from 5 to 1000contiguous amino acid residues of a reference polypeptide, respectively.In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150,250, or 500 contiguous amino acid residues of a reference polypeptide.Fragments may be preferentially selected from certain regions of amolecule. The term “at least a fragment” encompasses the full lengthpolypeptide.

Variants comprising insertions or additions relative to a referenceamino acid sequence of a peptide, polypeptide, or protein arecontemplated herein. The words “insertion” and “addition” refer tochanges in an amino acid or sequence resulting in the addition of one ormore amino acid residues. An insertion or addition may refer to 1, 2, 3,4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acidresidues.

Fusion proteins also are contemplated herein. A “fusion protein” refersto a protein formed by the fusion of at least one peptide, polypeptide,or protein or variant thereof as disclosed herein to at least oneheterologous protein peptide, polypeptide, or protein (or fragment orvariant thereof). The heterologous protein(s) may be fused at theN-terminus, the C-terminus, or both termini of the peptides or variantsthereof.

“Homology” refers to sequence similarity or, interchangeably, sequenceidentity, between two or more polypeptide sequences. Homology, sequencesimilarity, and percentage sequence identity may be determined usingmethods in the art and described herein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of residue matchesbetween at least two polypeptide sequences aligned using a standardizedalgorithm. Methods of polypeptide sequence alignment are well-known.Some alignment methods take into account conservative amino acidsubstitutions. Such conservative substitutions, explained in more detailabove, generally preserve the charge and hydrophobicity at the site ofsubstitution, thus preserving the structure (and therefore function) ofthe polypeptide. Percent identity for amino acid sequences may bedetermined as understood in the art. (See, e.g., U.S. Pat. No.7,396,664, which is incorporated herein by reference in its entirety). Asuite of commonly used and freely available sequence comparisonalgorithms is provided by the National Center for BiotechnologyInformation (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul,S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available fromseveral sources, including the NCBI, Bethesda, Md., at its website. TheBLAST software suite includes various sequence analysis programsincluding “blastp,” that is used to align a known amino acid sequencewith other amino acids sequences from a variety of databases.

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber (e.g., any of SEQ ID NOs:1-10), or may be measured over a shorterlength, for example, over the length of a fragment taken from a larger,defined polypeptide sequence, for instance, a fragment of at least 15,at least 20, at least 30, at least 40, at least 50, at least 70 or atleast 150 contiguous residues. Such lengths are exemplary only, and itis understood that any fragment length supported by the sequences shownherein, in the tables, figures or Sequence Listing, may be used todescribe a length over which percentage identity may be measured.

A “variant” of a particular polypeptide sequence may be defined as apolypeptide sequence having at least 50% sequence identity to theparticular polypeptide sequence over a certain length of one of thepolypeptide sequences using blastp with the “BLAST 2 Sequences” toolavailable at the National Center for Biotechnology Information'swebsite. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250). Such a pair of polypeptides may show,for example, at least 60%, at least 70%, at least 80%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% or greatersequence identity over a certain defined length of one of thepolypeptides. A “variant” may have substantially the same functionalactivity as a reference polypeptide (e.g., glycosylase activity or otheractivity). “Substantially isolated or purified” amino acid sequences arecontemplated herein. The term “substantially isolated or purified”refers to amino acid sequences that are removed from their naturalenvironment, and are at least 60% free, preferably at least 75% free,and more preferably at least 90% free, even more preferably at least 95%free from other components with which they are naturally associated.Variant polypeptides as contemplated herein may include variantpolypeptides of any of SEQ ID NOs:1-10).

As used herein, “translation template” refers to an RNA product oftranscription from an expression template that can be used by ribosomesto synthesize polypeptides or proteins.

The term “reaction mixture,” as used herein, refers to a solutioncontaining reagents necessary to carry out a given reaction. A reactionmixture is referred to as complete if it contains all reagents necessaryto perform the reaction. Components for a reaction mixture may be storedseparately in separate container, each containing one or more of thetotal components. Components may be packaged separately forcommercialization and useful commercial kits may contain one or more ofthe reaction components for a reaction mixture.

The steps of the methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The steps may be repeated or reiterated anynumber of times to achieve a desired goal unless otherwise indicatedherein or otherwise clearly contradicted by context.

Preferred aspects of this invention are described herein, including thebest mode known to the inventors for carrying out the invention.Variations of those preferred aspects may become apparent to those ofordinary skill in the art upon reading the foregoing description. Theinventors expect a person having ordinary skill in the art to employsuch variations as appropriate, and the inventors intend for theinvention to be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

Cell-Free Protein Synthesis (CFPS)

The strains and systems disclosed herein may be applied to cell-freeprotein synthesis methods as known in the art. See, for example, U.S.Pat. Nos. 4,496,538; 4,727,136; 5,478,730; 5,556,769; 5,623,057;5,665,563; 5,679,352; 6,168,931; 6,248,334; 6,531,131; 6,869,774;6,994,986; 7,118,883; 7,189,528; 7,338,789; 7,387,884; 7,399,610;8,703,471; and 8,999,668. See also U.S. Published Application Nos.2015-0259757, 2014-0295492, 2014-0255987, 2014-0045267, 2012-0171720,2008-0138857, 2007-0154983, 2005-0054044, and 2004-0209321. See alsoU.S. Published Application Nos. 2005-0170452; 2006-0211085;2006-0234345; 2006-0252672; 2006-0257399; 2006-0286637; 2007-0026485;2007-0178551. See also Published PCT International Application Nos.2003/056914; 2004/013151; 2004/035605; 2006/102652; 2006/119987; and2007/120932. See also Jewett, M. C., Hong, S. H., Kwon, Y. C., Martin,R. W., and Des Soye, B. J. 2014, “Methods for improved in vitro proteinsynthesis with proteins containing non standard amino acids,” U.S.Patent Application Ser. No. 62/044,221; Jewett, M. C., Hodgman, C. E.,and Gan, R. 2013, “Methods for yeast cell-free protein synthesis,” U.S.Patent Application Ser. No. 61/792,290; Jewett, M. C., J. A. Schoborg,and C. E. Hodgman. 2014, “Substrate Replenishment and Byproduct RemovalImprove Yeast Cell-Free Protein Synthesis,” U.S. Patent Application Ser.No. 61/953,275; and Jewett, M. C., Anderson, M. J., Stark, J. C.,Hodgman, C. E. 2015, “Methods for activating natural energy metabolismfor improved yeast cell-free protein synthesis,” U.S. Patent ApplicationSer. No. 62/098,578. See also Guarino, C., & DeLisa, M. P. (2012). Aprokaryote-based cell-free translation system that efficientlysynthesizes glycoproteins. Glycobiology, 22(5), 596-601. The contents ofall of these references are incorporated in the present application byreference in their entireties.

In certain exemplary embodiments, one or more of the methods describedherein are performed in a vessel, e.g., a single, vessel. The term“vessel,” as used herein, refers to any container suitable for holdingon or more of the reactants (e.g., for use in one or more transcription,translation, and/or glycosylation steps) described herein. Examples ofvessels include, but are not limited to, a microtitre plate, a testtube, a microfuge tube, a beaker, a flask, a multi-well plate, acuvette, a flow system, a microfiber, a microscope slide and the like.

In certain exemplary embodiments, physiologically compatible (but notnecessarily natural) ions and buffers are utilized for transcription,translation, and/or glycosylation, e.g., potassium glutamate, ammoniumchloride and the like. Physiological cytoplasmic salt conditions arewell-known to those of skill in the art.

The strains and systems disclosed herein may be applied to cell-freeprotein methods in order to prepare glycosylated macromolecules (e.g.,glycosylated peptides, glycosylated proteins, and glycosylated lipids).Glycosylated proteins that may be prepared using the disclosed strainsand systems may include proteins having N-linked glycosylation (i.e.,glycans attached to nitrogen of asparagine and/or arginine side-chains)and/or O-linked glycosylation (i.e., glycans attached to the hydroxyloxygen of serine, threonine, tyrosine, hydroxylysine, and/orhydroxyproline). Glycosylated lipids may include O-linked glycans via anoxygen atom, such as ceramide.

The glycosylated macromolecules disclosed herein may include unbranchedand/or branched sugar chains composed of monomers as known in the artsuch as, but not limited to, glucose (e.g., β-D-glucose), galactose(e.g., β-D-galactose), mannose (e.g., β-D-mannose), fucose (e.g.,α-L-fucose), N-acetyl-glucosamine (GlcNAc), N-acetyl-galactosamine(GalNAc), neuraminic acid, N-acetylneuraminic acid (i.e., sialic acid),and xylose, which may be attached to the glycosylated macromolecule,growing glycan chain, or donor molecule (e.g., a donor lipid and/or adonor nucleotide) via respective glycosyltransferases (e.g.,oligosaccharyltransferases, GlcNAc transferases, GalNAc transferases,galactosyltransferases, and sialyltransferases). The glycosylatedmacromolecules disclosed herein may include glycans as known in the art.

The disclosed cell-free protein synthesis systems may utilize componentsthat are crude and/or that are at least partially isolated and/orpurified. As used herein, the term “crude” may mean components obtainedby disrupting and lysing cells and, at best, minimally purifying thecrude components from the disrupted and lysed cells, for example bycentrifuging the disrupted and lysed cells and collecting the crudecomponents from the supernatant and/or pellet after centrifugation. Theterm “isolated or purified” refers to components that are removed fromtheir natural environment, and are at least 60% free, preferably atleast 75% free, and more preferably at least 90% free, even morepreferably at least 95% free from other components with which they arenaturally associated.

Genetically Modified Organisms

In some embodiments of the disclosed methods, a genetically modifiedorganism is utilized. In some embodiments, the genetically modifiedprokaryote is a genetically modified strain of Escherichia coli or anyother prokaryote suitable for preparing a lysate for CFGpS. Optionally,the modified strain of Escherichia coli is derived from rEc.C321.Preferably, the modified strain includes genomic modifications (e.g.,deletions of genes rendering the genes inoperable) that preferablyresult in lysates capable of high-yielding cell-free protein synthesis.Also, preferably, the modified strain includes genomic modification(e.g., deletions of genes rendering the genes inoperable) thatpreferably result in lysates comprising sugar precursors forglycosylation at relatively high concentrations (e.g., in comparison toa strain not having the genomic modification). In some embodiments, alysate prepared from the modified strain comprises sugar precursors at aconcentration that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 150%, 200%, or higher than a lysate prepared from a strain that isnot modified.

In some embodiments, the modified strain includes a modification thatresults in an increase in the concentration of a monosaccharide utilizedin glycosylation (e.g., glucose, mannose, N-acetyl-glucosamine (GlcNAc),N-acetyl-galactosamine (GalNAc), galactose, sialic acid, neuraminicacid, fucose). As such, the modification may inactivate an enzyme thatmetabolizes a monosaccharide or polysaccharide utilized inglycosylation. In some embodiments, the modification inactivates adehydratase or carbon-oxygen lyase enzyme (EC 4.2) (e.g., via a deletionof at least a portion of the gene encoding the enzyme). In particular,the modification may inactivate a GDP-mannose 4,6-dehydratase (EC4.2.1.47). When the modified strain is E. coli, the modification mayinclude an inactivating modification in the gmd gene (e.g., via adeletion of at least a portion of the gmd gene). The sequence of the E.coli gmd gene is provided herein as SEQ ID NO:1 and the amino acidsequence of E. coli GDP-mannose 4,6-dehydratase is provided as SEQ IDNO:2.

In some embodiments, the modified strain includes a modification thatinactivates an enzyme that is utilized in the glycosyltransferasepathway. In some embodiments, the modification inactivates anoligosaccharide ligase enzyme (e.g., via a deletion of at least aportion of the gene encoding the enzyme). In particular, themodification may inactivate an O-antigen ligase that optionallyconjugates an O-antigen to a lipid A core oligosaccharide. Themodification may include an inactivating modification in the waaL gene(e.g., via a deletion of at least a portion of the waaL gene). Thesequence of the E. coli waaL gene is provided herein as SEQ ID NO:3 andthe amino acid sequence of E. coli O-antigen ligase is provided as SEQID NO:4.

In some embodiments, the modified strain includes a modification thatinactivates a dehydratase or carbon-oxygen lyase enzyme (e.g., via adeletion of at least a portion of the gene encoding the enzyme) and alsothe modified strain includes a modification that inactivates anoligosaccharide ligase enzyme (e.g., via a deletion of at least aportion of the gene encoding the enzyme). The modified strain mayinclude an inactivation or deletion of both gmd and waaL.

In some embodiments, the modified strain may be modified to express oneor more orthogonal or heterologous genes. In particular, the modifiedstrain may be genetically modified to express an orthogonal orheterologous gene that is associated with glycoprotein synthesis such asa glycosyltransferase (GT) which is involved in the lipid-linkedoligosaccharide (LLO) pathway. In some embodiments, the modified strainmay be modified to express an orthogonal or heterologousoligosaccharyltransferase (EC 2.4.1.119) (OST).Oligosaccharyltransferases or OSTs are enzymes that transferoligosaccharides from lipids to proteins.

In particular, the modified strain may be genetically modified toexpress an orthogonal or heterologous gene in a glycosylation system(e.g., an N-linked glycosylation system and/or an O-linked glycosylationsystem). The N-linked glycosylation system of Campylobacter jejuni hasbeen transferred to E. coli. (See Wacker et al., “N-linked glycosylationin Campylobacter jejuni and its functional transfer into E. coli,”Science 2002, Nov. 29; 298(5599):1790-3, the content of which isincorporated herein by reference in its entirety). In particular, themodified strain may be modified to express one or more genes of the pgllocus of C. jejuni or one or more genes of a homologous pgl locus. Thegenes of the pgl locus include pglG, pglF, pglE, wlaJ, pglD, pglC, pglA,pglB, pglJ, pglI, pglH, pglK, and gne, and are used to synthesizelipid-linked oligosaccharides (LLOs) and transfer the oligosaccharidemoieties of the LLOs to a protein via an oligosaccharyltransferase.

Suitable orthogonal or heterologous oligosaccharyltransferases (OST)which may be expressed in the genetically modified strains may includeCampylobacter jejuni oligosaccharyltransferase Pg1B. The gene for the C.jejuni OST is referred to as pglB, which sequence is provided as SEQ IDNO:5 and the amino acid sequence of C. jejuni Pg1B is provided as SEQ IDNO:6. Pg1B catalyzes transfer of an oligosaccharide to a D/E-Y-N-X-S/Tmotif (Y, X≠P) present on a protein.

Crude cell lysates may be prepared from the modified strains disclosedherein. The crude cell lysates may be prepared from different modifiedstrains as disclosed herein and the crude cell lysates may be combinedto prepare a mixed crude cell lysate. In some embodiments, one or morecrude cell lysates may be prepared from one or more modified strainsincluding a genomic modification (e.g., deletions of genes rendering thegenes inoperable) that preferably result in lysates comprising sugarprecursors for glycosylation at relatively high concentrations (e.g., incomparison to a strain not having the genomic modification). In someembodiments, one or more crude cell lysates may be prepared from one ormore modified strains that have been modified to express one or moreorthogonal or heterologous genes or gene clusters that are associatedwith glycoprotein synthesis. Preferably, the crude cell lysates or mixedcrude cell lysates are enriched in glycosylation components, such aslipid-linked oligosaccharides (LLOs), glycosyltransferases (GTs),oligosaccharyltransferases (OSTs), or any combination thereof. Morepreferably, the crude cell lysates or mixed crude cell lysates areenriched in Man₃GlcNAc₂ LLOs representing the core eukaryotic glycanand/or Man₃GlcNAc₄Gal₂Neu₅Ac₂ LLOs representing the fully sialylatedhuman glycan.

The disclosed crude cell lysates may be used in cell-free glycoproteinsynthesis (CFGpS) systems to synthesize a variety of glycoproteins. Theglycoproteins synthesized in the CFGpS systems may include prokaryoticglycoproteins and eukaryotic proteins, including human proteins. TheCFGpS systems may be utilized in methods for synthesizing glycoproteinsin vitro by performing the following steps using the crude cell lysatesor mixtures of crude cell lysates disclosed herein: (a) performingcell-free transcription of a gene for a target glycoprotein; (b)performing cell-free translation; and (c) performing cell-freeglycosylation. The methods may be performed in a single vessel ormultiple vessels. Preferably, the steps of the synthesis method may beperformed using a single reaction vessel. The disclosed methods may beused to synthesis a variety of glycoproteins, including prokaryoticglycoproteins and eukaryotic glycoproteins.

In some embodiments, the engineered modified organism may be engineeredto express a detoxified from of lipid A and/or reduced amounts of lipidA. As such, the engineered modified organisms can be utilized to preparelysates that are less toxic than the unmodified organisms (i.e.wild-type organisms). In some embodiments, the engineered modifiedorganism may be engineered to include a deletion in the IpxM gene and/ormay be engineered to express the LpxE gene product (e.g., the F.tularensis LpxE gene product). As such, the engineered modifiedorganisms can be utilized as source strains to prepare lysates that aredetoxified via deletion of lpxM and/or expression of the F. tularensisLpxE gene product in the source strains.

Bioconjugate Vaccine Production

While protein-glycan coupling technology (PGCT) represents a simplifiedand cost-effective strategy for bioconjugate vaccine production, it hasthree main limitations. First, process development timelines,glycosylation pathway design-build-test (DBT) cycles, and bioconjugateproduction are all limited by cellular growth. Second, it has not yetbeen shown whether FDA-approved carrier proteins, such as the toxinsfrom Clostridium tetani and Corynebacterium diptheriae, are compatiblewith N-linked glycosylation in living E. coli. Third, select non-nativeglycans are known to be transferred with low efficiency by the C. jejunioligosaccharyltransferase (OST), Pg1B.

A modular, in vitro platform for production of bioconjugates has thepotential to address all of these limitations. Here, we demonstrate thatbioconjugates against pathogenic strains of Franciscella tualrensis andEscherichia coli can be produced through coordinated in vitrotranscription, translation, and N-glycosylation in cell-freeglycoprotein synthesis (CFGpS) reactions lasting just 20 hours. Thissystem has the potential to reduce process development and distributiontimelines for novel antibacterial vaccines from weeks to days. Further,because of the modular nature of the CFGpS platform and the fact thatcell-free systems have demonstrated advantages for production ofmembrane proteins compared to living cells, this method could be readilyapplied to produce bioconjugates using FDA-approved carrier proteins,such as the Clostridium tetani and Corynebacterium diptheriae toxins,which are membrane localized. This could be accomplished simply bysupplying plasmid encoding these carrier proteins to CFGpS reactions.Additionally, because of the modular nature of CFGpS, the in vitroapproach could be used to prototype other natural or engineered homologsof the archetypal C. jejuni OST to identify candidate OSTs with improvedefficiency for transfer of O-antigen LLOs of interest. This can beaccomplished by enriching lysates with OSTs of interest and mixing themwith LLO lysates in mixed lysate CFGpS reactions, as we have describedpreviously. (See WO 2017/117539, the content of which is incorporatedherein by reference in its entirety). Additionally, the methods can beperformed using lysates prepared from microorganism that have beengenetically modified to produce reduced amounts of endotoxin orde-toxified endotoxin, as required by FDA safety guidelines for vaccineproduction.

For example, we demonstrate that cell-free bioconjugate synthesislysates can be detoxified by genetically engineering a host strain suchthat the structure of lipid A produced by the host strain exhibits toreduced toxicity. In some embodiments, the disclosed lysates preparedfrom genetically engineered host strains have a concentration ofendotoxin units (EU) that is less than about 200,000 EU/mL, 180,000EU/mL, 160,000 EU/mL, 140, 000 EU/mL, 120, 000 EU/mL, 100,000 EU/mL,80,000 EU/mL, 60,000 EU/mL, 40,000 EU/mL, 20,000 EU/mL, 10,000 EU/mL,8,000 EU/mL, 6,000 EU/mL, 4,000 EU/mL, 2,000 EU/mL, 1,000 EU/mL, 800EU/mL, 600 EU/mL, 400 EU/mL, 200 EU/mL, 100 EU/mL, 80 EU/mL, 60 EU/mL,40 EU/mL, 20 EU/mL, 10 EU/mL, 8 EU/mL, 6 EU/mL, 4 EU/mL 2 EU/mL 1 EU/mL,0.9 EU/mL, 0.8 EU/mL 0.7 EU/mL, 0.6 EU/mL, 0.5 EU/mL, 0.4 EU/mL, 0.3EU/mL, 0.2 EU/mL, 0.1 EU/mL, 0.05 EU/mL, or within a concentration rangebounded by any of these values (e.g., within a range of about0.3-180,000 EU/mL). The disclosed advances make it possible, for thefirst time, to produce and glycosylate authentic FDA-approved carrierproteins in detoxified lysates. As such, the detoxified lysates can beused in cell-free reaction mixtures to produce N-glycosylated carrierproteins as disclosed herein and the cell-free reaction mixtures can bedirectly administered (e.g., via injection) into a subject in needthereof (e.g., in order to induce an immune response which optionallyprotects the subject from infection by a microorganism that expressesthe polysaccharide of the N-glycosylated carrier proteins) withoutrequiring that the N-glycosylated carrier proteins first be purifiedfrom the cell-free reaction mixtures prior to being administered to thesubject (i.e., the cell-free reaction mixture comprising theN-glycosylated carrier proteins may be administered directly to thesubject).

Finally, we demonstrate that cell-free bioconjugate synthesis reactionscan be lyophilized and retain bioconjugate synthesis capability,demonstrating the potential of the CFGpS system for on-demand, portable,and low cost production or development efforts for novel vaccines. Thisnovel method for in vitro bioconjugate vaccine production hasdemonstrated advantages for rapid, modular, and portable vaccineprototyping and production compared to existing methods. Our technologyenables rapid production of bioconjugate vaccines directed against auser-specified bacterial target for therapeutic development orfundamental research.

The present inventors are not aware of any prokaryotic cell-free systemwith the capability to produce glycoproteins or bioconjugate vaccines.There are commercial eukaryotic cell lysate systems for cell-freeglycoprotein production, but these systems do not involve overexpressionof orthogonal glycosylation machinery and do not enable modular,user-specified glycosylation in the way our system can. Additionally,these systems contain wild-type lipid A structures that would have to beremoved from the vaccine-producing reaction to meet FDA regulationsregarding endotoxin content in vaccine preparations. For these reasons,the disclosed technology is advantageous over technology in the priorart.

Finally, the cell-free platform disclosed here uniquely enablesproduction of FDA-approved vaccine carriers at high soluble yields.Typically, production of the inventors' knowledge, this is the firstreport of recombinant production of tetanus toxin, which is commerciallyharvested from the pathogen Clostridium tetani. For these reasons, thedisclosed technology has unique advantages for the production andconjugation of FDA-approved conjugate vaccines and thus has significantcommercial promise.

The presently disclosed method for in vitro bioconjugate vaccineproduction has demonstrated advantages for rapid, modular, and portablevaccine prototyping and production compared to existing methods. Thedisclosed methods address limitations of existing production approaches,making the disclosed methods an attractive alternative or complementarystrategy for antibacterial vaccine production. In light of the growinghealthcare concerns caused by antibiotic-resistant bacterial infections,the disclosed methods have the potential to be extremely valuable fordevelopment, production, and distribution of novel vaccines againstdiverse pathogenic bacterial strains. Advantages of the disclosedmethods include: on demand expression of bioconjugate vaccines;prototyping novel bioconjugate vaccine candidates; prototyping novelbioconjugate vaccine production pathways; distribution of bioconjugatevaccines to resource-poor settings.

In summary, we disclose the first prokaryotic cell-free system capableof coordinated, cell-free transcription, translation, and glycosylationof glycoprotein vaccines. The disclosed system enables production ofbioconjugate vaccines in a short period of time (e.g., less than 24, 20,16, 12, 8, 6, 4, or 2 hours). The modularity of the system enables rapidprototyping of novel glycosylation pathways and vaccine candidates withvarious carrier proteins. Suitable carriers may include but are notlimited to Haemophilus influenzae protein D (PD) (SEQ ID NO: 7),Neisseria meningitidis porin protein (PorA) (SEQ ID NO: 8),Corynebacterium diphtherias toxin (CRM197) (SEQ ID NO: 9), Clostridiumtetani toxin (TT) (SEQ ID NO: 10), and Escherichia coli maltose bindingprotein, or variants thereof. The components and products of the systemmaybe lyophilized, which enables the potential for broad and rapiddistribution of vaccine production technology. The disclosed systemreduces the time required to produce bioconjugates in a prokaryotic celllysate from weeks to days, which could provide competitive advantage incommercialization of the technology.

Vaccine Formulations

The N-glycosylated carrier proteins disclosed herein may be formulatedas antigenic compositions and/or vaccine composition for administrationto a subject in need thereof. Such compositions can be formulated and/oradministered in dosages and by techniques well known to those skilled inthe medical arts taking into consideration such factors as the age, sex,weight, and condition of the particular patient, and the route ofadministration. The compositions may include pharmaceutical carriers,diluents, or excipients as known in the art. Further, the compositionsmay include preservatives (e.g., anti-microbial or anti-bacterial agentssuch as benzalkonium chloride) or adjuvants.

The compositions disclosed herein may be delivered via a variety ofroutes. Typical delivery routes include parenteral administration (e.g.,intradermal, intramuscular or subcutaneous delivery). Other routesinclude oral administration, intranasal, intravaginal, intrarectalroutes. The compositions may be formulated for intranasal or pulmonarydelivery. Formulations of the pharmaceutical compositions may includeliquid formulations (e.g., for oral, nasal, anal, vaginal, etcadministration, including suspensions, syrups or elixirs) andpreparations for parenteral, subcutaneous, intradermal, intramuscular orintravenous administration (e.g., injectable administration) such assterile suspensions or emulsions.

The disclosed antigenic compositions and vaccine compositions optionallymay include additional agents for inducing and/or potentiating an immuneresponse such as adjuvants and/or may be administered with an adjuvant.The term “adjuvant” refers to a compound or mixture that enhances animmune response. An adjuvant can serve as a tissue depot that slowlyreleases the antigen and also as a lymphoid system activator thatnon-specifically enhances the immune response. Examples of adjuvantswhich may be utilized in the disclosed compositions include but are notlimited to, co-polymer adjuvants (e.g., Pluronic L121® brand poloxamer401, CRL1005, or a low molecular weight co-polymer adjuvant such asPolygen® adjuvant), poly (I:C), R-848 (a Th1-like adjuvant), resiquimod,imiquimod, PAM3CYS, aluminum phosphates (e.g., AlPO₄), loxoribine,potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin)and Corynebacterium parvum, CpG oligodeoxynucleotides (ODN), choleratoxin derived antigens (e.g., CTA1-DD), lipopolysaccharide adjuvants,complete Freund's adjuvant, incomplete Freund's adjuvant, saponin (e.g.,Quil-A), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil or hydrocarbon emulsions in water (e.g., MF59 available fromNovartis Vaccines or Montanide ISA 720), keyhole limpet hemocyanins, anddinitrophenol.

The disclosed antigenic compositions and vaccine compositions may beadministered under a prime-boost vaccination regimen. As used herein, a“prime-boost vaccination regimen” refers to a regimen in which a subjectis administered a first composition one or more times (e.g., two orthree times with about 2, 3, or 4 weeks between administrations) andthen after a determined period of time (e.g., about 2 weeks, about 4weeks, about 2 months, about 3 months, about 4 months, about 5 months,about 6 months, or longer), the subject is administered a secondcomposition. The second composition may also be administered more thanonce, with at least 2, 3, or 4 weeks between administrations. The firstand second compositions may be the same or different.

Illustrative Embodiments

The following embodiments are illustrative and are not intended to limitthe scope of the claimed subject matter.

Embodiment 1. A method for synthesizing a N-glycosylated recombinantprotein carrier which optionally may be utilized for preparing abioconjugate vaccine, the method comprising performing coordinatedtranscription, translation, and N-glycosylation of the recombinantprotein carrier in a cell-free reaction mixture thereby providing theN-glycosylated recombinant protein carrier which may be utilized as thebioconjugate vaccine, wherein the N-glycosylated recombinant proteincarrier comprises: (i) a consensus sequence (which optionally isinserted in the protein carrier), N-X-S/T, wherein X may be any naturalor unnatural amino acid except proline; and (ii) at least one antigenicpolysaccharide from at least one bacterium N-linked to the recombinantprotein carrier, wherein the at least one antigenic polysaccharideoptionally is at least one bacterial O-antigen, optionally from one ormore strains of E. coli or Franciscella tularensis; and the bioconjugatevaccine optionally may include an adjuvant, optionally wherein thecell-free reaction mixture comprises a lysate prepared from anengineered bacterial strain that expresses a detoxified form of lipid Aor lower amounts of lipid A relative to a wild-type strain.

Embodiment 2. The method of embodiment 1, wherein the carrier protein isan engineered variant of E. coli maltose binding protein (MBP).

Embodiment 3. The method of embodiment 1, wherein the carrier protein isselected from a detoxified variant of the toxin from Clostridium tetaniand a detoxified variant of the toxin from Corynebacterium diptheriae

Embodiment 4. The method of embodiment 1, wherein the carrier protein isselected from Haemophilus influenzae protein D (PD) and Neisseriameningitidis porin protein (PorA), and variants thereof.

Embodiment 5. The method of any of the foregoing embodiments, whereinthe method utilizes an oligosaccharyltransferase (OST) which is anaturally occurring bacterial homolog of C. jejuni Pg1B.

Embodiment 6. The method of any of embodiments 1-4, wherein the methodutilizes an OST that is an engineered variant of C. jejuni Pg1B.

Embodiment 7. The method of any of embodiments 1-4, wherein the methodutilizes an OST that is a naturally occurring archaeal OST.

Embodiment 8. The method of any of embodiments 1-4, wherein the methodutilizes an OST which is a naturally occurring single-subunit eukaryoticOST, such as those found in Trypanosoma bruceii.

Embodiment 9. The method of any of the foregoing embodiments, whichutilizes a lysate prepared from a detoxified strain of E. coli, forexample, a strain of E. coli which is deficient in IpxM (i.e., AIpxM)and/or a strain of E. coli that has been engineered to express the LpxEgene product (e.g., the F. tularensis LpxE gene product). (See, e.g.,Needham et al., PNAS, 110(4), 1464-1469 (2013); and Brito and Singh, J.Pharma. Sci., 100(1), 34-37 (2011); the contents of which areincorporated herein by reference in their entireties).

Embodiment 9. A method for crude cell lysate preparation in whichorthogonal genes or gene clusters are expressed in a source strain forthe crude cell lysate, which results in lysates enriched withglycosylation components (lipid-linked oligosaccharides (LLOs),oligosaccharyltransferases (OSTs), and/or both LLOs and OSTs), andoptionally which results in a separate lysate enriched with LLOs (e.g.,LLOs associated with O-antigen) and a separate lysate enriched with OSTs(e.g., for which the LLOs are a substrate), and optionally combining theseparate lysates to perform cell-free protein synthesis of a carrierprotein which is glycosylated with the glycan component of the LLOs viathe OST's enzyme activity, and further optionally purifying theglycosylated carrier protein and optionally administering theglycosylated carrier protein as an immunogen.

Embodiment 10. The method of embodiment 9, in which the source strainoverexpresses a gene encoding an oligosaccharyltransferase (OST).

Embodiment 11. The method of embodiment 9 or 10, in which the sourcestrain overexpresses a synthetic glycosyltransferase pathway, resultingin the production of O-antigens, optionally O-antigens from F.tularensis Schu S4 lipid-linked oligosaccharides (FtLLOs).

Embodiment 12. The method of embodiment 9 or 10, in which the sourcestrain overexpresses a synthetic glycosyltransferase pathway, resultingin the production of O-antigens, optionally O-antigens fromenterotoxigenic E. coli O78 lipid-linked oligosaccharides (EcO78LLOs)

Embodiment 13. The method of any of embodiments 9-12, in which thesource strain overexpresses a glycosyltransferase pathway and an OST,resulting in the production of LLOs and OST.

Embodiment 14. The method of embodiment 9 or 10, in which the sourcestrain overexpresses an O-antigen glycosyltransferase pathway from apathogenic bacterial strain, resulting in the production of O-antigenlipid-linked oligosaccharides (LLOs).

Embodiment 15. A method for cell-free production of a bioconjugatevaccine that involves mixing crude cell lysates (e.g., any of the crudecell lysates of embodiments 9-14).

Embodiment 16. The method of embodiment 15, in which the bioconjugatevaccine comprises an immunogenic carrier that is a protein or a peptide.

Embodiment 17. The method of embodiment 15, in which the bioconjugatevaccine comprises an immunogenic carrier that is a protein or peptidecomprising a protein or peptide thereof selected form Haemophilusinfluenzae protein D (PD), Neisseria meningitidis porin protein (PorA),Corynebacterium diphtherias toxin (CRM197), Clostridium tetani toxin(TT), and Escherichia coli maltose binding protein, and variantsthereof.

Embodiment 18. The method of any of embodiments 1-17 in which thecomponents of the method may be lyophilized and retain bioconjugatesynthesis capability when rehydrated.

Embodiment 19. The method of any of embodiments 15-18 where the goal ison-demand vaccine production.

Embodiment 20. The method of any of embodiments 15-19 where the goal isvaccine production in resource-limited settings.

Embodiment 21. A kit for synthesizing a N-glycosylated carrier proteinin vitro, the kit comprising one or more of the following components:(i) a first component comprising a cell lysate that comprises anorthogonal oligosaccharyltransferase (OST); (ii) a second componentcomprising a cell lysate that comprises an O-antigen (e.g., lipid-linkedoligosaccharides (LLOs) comprising O-antigen; (iii) a third componentcomprising a transcription template and optionally a polymerase forsynthesizing an mRNA from the transcription template encoding a carrierprotein, the carrier protein comprising an inserted and/or a naturallyoccurring consensus sequence, N-X-S/T, wherein X may be any natural orunnatural amino acid except proline.

Embodiment 22. The kit of embodiment 21, wherein one or more of thefirst component, the second component, and the third component arelyophilized and retain biological activity when rehydrated.

Embodiment 23. The kit of embodiment 21 or 22, wherein the firstcomponent cell lysate is produced from a source strain (e.g., E. coli)that overexpresses a gene encoding the orthogonal OST (e.g. C. jejuniPg1B).

Embodiment 24. The kit of any of embodiments 21-23, wherein the secondcomponent cell lysate is produced from a source strain thatoverexpresses a synthetic glycosyltransferase pathway (e.g., thebiosynthetic machinery to produce the Franciscella tularensis Schu S4O-antigen (FtLLOs lysate) or the biosynthetic machinery to produce theenterotoxigenic E. coli O78 lipid-linked oligosaccharides (EcO78LLOslysate).

Embodiment 25. A method for cell-free production of a glycoprotein whichoptionally may be a bioconjugate suitable for use as a vaccine, themethod comprising: (a) mixing a first cell lysate comprising anorthogonal oligosaccharyltransferase (OST) and a second cell lysate thatcomprises an O-antigen (e.g., as lipid-linked oligosaccharides (LLOs))to prepare a cell-free protein synthesis reaction; (b) transcribing andtranslating a carrier protein in the cell-free protein synthesisreaction (e.g., optionally by adding a transcription template for thecarrier protein and/or a polymerase to the cell-free protein synthesisreaction), the carrier protein comprising an inserted and/or naturallyoccurring consensus sequence, N-X-S/T, wherein X may be any natural orunnatural amino acid except proline; and (c) glycosylating the carrierprotein in the cell-free protein synthesis reaction with the bacterialO-antigen.

Embodiment 25. The method of embodiment 24, wherein the second celllysate comprises the O-antigen as part of lipid-linked oligosaccharides(LLOs).

Embodiment 26. The method of embodiment 24 or 25, further comprisingformulating the glycoprotein as a vaccine composition optionallyincluding an adjuvant.

Embodiment 27. A vaccine prepared by any of the foregoing methods and/orkits and/or a vaccine comprising a component present in or prepared byany of the foregoing methods and/or kits.

Embodiment 28. A vaccination method comprising administering the vaccineof embodiment 27 to a subject in need thereof.

EXAMPLES

The following Examples are illustrative and are not intended to limitthe scope of the claimed subject matter.

Example 1 On-Demand, Cell-Free Biomanufacturing of Conjugate Vaccine atthe Point-of Care

Abstract

Conjugate vaccines are highly effective against bacterial pathogens withextremely rare instances of resistance, representing a powerful tool torespond to the emerging threat of drug resistant bacteria. However,current technology for making conjugate vaccines is technically complexand relies on living cells, which necessitates highly centralizedmanufacturing, specialized equipment, lengthy development timelines, andcold-chain distribution. Here, we describe a modular technology forportable, on-demand in vitro bioconjugate vaccine expression (iVAX),which combines cell-free protein synthesis of licensed vaccine carrierproteins with N-linked glycosylation of bacterial polysaccharideantigens in freeze-dried lysates from detoxified, nonpathogenicEscherichia coli. Upon rehydration, iVAX technology can synthesizesingle doses of bioconjugate vaccines against diverse bacterialpathogens in one hour, including Franciscella tularensis subsp.tularensis (type A) strain Schu S4 and pathogenic E. coli strains O78and O7 Immunization of BALB/c mice with cell-free synthesized vaccineselicited pathogen-specific antibodies at significantly higher levelscompared to the same vaccine molecules produced using glycoengineeredbacteria. The iVAX platform promises to enable rapid development of newbioconjugate vaccines with increased access throughrefrigeration-independent distribution and on-demand, decentralizedproduction.

Introduction

For decades, vaccines have been an important pillar in preventativemedicine, providing protection against a wide array of disease-causingpathogens by induction of humoral and/or cellular immunity. Conjugatevaccines are among the safest and most effective methods for preventinglife-threatening bacterial infections [1] and have been shown to reducethe rate of emergence of drug resistant strains [2]. Conjugate vaccinesare composed of an immunogenic (CD4+ T-cell dependent antigen) proteincarrier chemically conjugated to a polysaccharide antigen from thesurface of the target bacterium, typically capsular polysaccharide(CPS)- or lipopolysaccharide (LPS)-derived antigens. Theseprotein-polysaccharide conjugates induce immune responses directedagainst the bacterial polysaccharide antigen characterized bypolysaccharide-specific IgM-to-IgG class switching, memory B celldevelopment, and long-lived T-cell memory [3-7]. As a result, conjugatevaccines have proven to be a highly efficacious and safe strategy forprotecting against virulent bacterial pathogens, including Haemophilusinfluenzae, Neisseria meningitidis, and Streptococcus pneumoniae [7-9],resulting in several marketed products and many others in clinicaldevelopment [3, 8].

Despite their effectiveness, conjugate vaccines are particularlychallenging to develop and distribute for several reasons. First, theconventional process to produce conjugate vaccines involvesbiosynthesis, purification, and chemical conjugation of thepolysaccharide and protein components, which is complex, costly, and lowyielding [10]. Moreover, in some cases, chemical conjugation cansignificantly alter the structure of polysaccharide, resulting in theloss of the protective epitope [11]. Second, biosynthesis of thepolysaccharide component typically requires large-scale cultivation ofpathogenic bacteria, which is accompanied by biosafety regulations andlimits the development of new conjugate vaccines to bacterial targetsthat are amenable to large-scale fermentation under normal laboratoryconditions. Third, as a result of process complexity and associatedbiosafety concerns, conjugate vaccine manufacturing takes place incentralized production facilities from which vaccines are distributedvia a refrigerated supply chain, which is critical to avoidprecipitation and significant loss of the pathogen-specific carbohydratecomponent upon both heating and freezing [10, 12, 13]. Accidentalheating and freezing in the cold chain result in significant vaccinespoilage [14] and, more broadly, the need to establish and maintain coldchain refrigeration creates economic and logistical challenges thatlimit the reach of vaccination campaigns, especially in the developingworld [12, 15].

As an alternative to chemical conjugation, it was recently demonstratedthat polysaccharide-protein conjugates can be made in Escherichia colivia protein-glycan coupling technology (PGCT) [16]. In this approach,engineered E. coli cells covalently attach heterologously expressed CPSor O-PS antigens to specific residues on carrier proteins in the E. coliperiplasm via an asparagine-linked (N-linked) glycosylation reactioncatalyzed by the Campylobacter jejuni oligosaccharyltransferase Pg1B(CjPg1B). To date, this technology has yielded a handful of vaccinecandidates, termed “bioconjugates” to highlight the in vivo productionprocess, including those directed against important human pathogensincluding Burkholderia pseudomallei [17], E. coli O121 [18], E. coliO157:H7 [19], Francisella tularensis [20, 21], and Staphylococcus aureus[22]. In all cases tested, the vaccines either stimulated serumbactericidal antibodies [19] or provided protection against pathogenchallenge [17, 20-22]. While PGCT can avoid some challenges associatedwith conventional conjugation vaccine manufacturing (e.g., biosafetyconcerns associated with growing pathogenic bacteria), bioconjugatevaccine production still relies on living bacterial cells. Thus, as withconventional conjugation approaches, bioconjugate vaccine productionremains subject to lengthy in vivo process development timelines and isdependent upon skilled operators and specialized equipment, whichaltogether necessitate centralized production facilities.

Cell-free protein synthesis (CFPS) is an emerging technology that usescell lysates, rather than living cells, to synthesize proteins in vitro[23]. CFPS has recently been used to enable rapid, low-cost, anddecentralized production of protein subunit vaccines [24, 25], proteintherapeutics [25], and diagnostics [26, 27]. Importantly, CFPStechnology (i) allows for shortened protein synthesis timelines, asrelevant yields of protein can be synthesized in vitro in just a fewhours, (ii) can be freeze-dried for transportation and storage atambient temperature and reconstituted by just adding water [24, 25], and(iii) circumvents biosafety concerns associated with the use of livingcells outside of a controlled laboratory setting. However, untilrecently, cell-free systems have been limited in their ability tosynthesize glycosylated proteins at relevant titers and in terms of therelatively small number of glycan structures that can be installed onproteins [28], preventing the synthesis of bioconjugate vaccines incell-free systems. We recently described a cell-free glycoproteinsynthesis (CFGpS) technology that enables one-pot, cell-free productionof glycosylated proteins [29]. CFGpS is a modular platform in whichacceptor protein, polysaccharide, and oligosaccharyltransferasecomponents can be rapidly interchanged to yield cell-free production ofstructurally diverse glycoproteins, including human glycoproteins andeukaryotic glycans [29]. However, it remains to be shown whether asimilar cell-free approach can be used for in vitro synthesis ofbioconjugate vaccines.

To address this gap, here we describe a cell-free platform for portable,on-demand in vitro bioconjugate vaccine expression (iVAX). iVAX enablesrapid, inexpensive, and portable production of safe and effectiveantibacterial vaccine candidates via complete cell-free biosynthesis andglycosylation of licensed carrier proteins with bacterial polysaccharideantigens (FIG. 1 ). iVAX was designed to have the following features.First, iVAX is fast. It can produce FDA-approved carrier proteins atlevels sufficient for individual doses in 1-2 h. Second, iVAX is robust,operating under a range of temperature conditions. Third, iVAX ismodular, being capable of efficient antigen conjugation of diverseO-antigen polysaccharides (O-PS), including the highly virulentFranciscella tularensis subsp. tularensis (type A) strain Schu S4,enterotoxigenic (ETEC) E. coli O78, and uropathogenic (UPEC) E. coli O7.Fourth, iVAX is shelf-stable, derived from freeze-dried cell-freereactions that operate in a just-add-water strategy. Fifth, iVAX issafe, leveraging lipid A remodeling that effectively avoids the highlevels of endotoxin that are present in non-engineered E. colimanufacturing platforms. We demonstrate that that anti-F. tularensisbioconjugates derived from freeze-dried, low-endotoxin iVAX reactionselicit pathogen-specific antibody responses in mice and outperform abioconjugate produced using the established PGCT approach in livingcells. Overall, this work represents an alternative approach tocentralized conjugate vaccine biomanufacturing which could be readilyadapted for developing vaccine candidates against many other bacterialpathogens and offers a fundamentally new way to deliver the protectivebenefits of existing vaccine technologies to both the developed anddeveloping world.

Results

In vitro synthesis of licensed vaccine carrier proteins. To demonstrateproof-of-principle for point-of-care conjugate vaccine production, wefirst set out to express a set of carrier proteins that are currentlyused in FDA-approved conjugate vaccines. Producing these carrierproteins in soluble conformations in vitro represented an importantbenchmark because their expression in living E. coli has provenchallenging, often requiring purification and refolding of insolubleproduct from inclusion bodies [30, 31], fusion of expression partnerssuch as maltose binding protein (MBP) to increase soluble expression[31, 32], or expression of protein fragments in favor of the full-lengthprotein [32]. In contrast, cell-free protein synthesis approaches haverecently shown promise for difficult-to-express proteins [33, 34]. Thecarrier proteins that we focused on here included nonacylated H.influenzae protein D (PD), the N. meningitidis porin protein (PorA), andgenetically detoxified variants of the Corynebacterium diphtheriae toxin(CRM197) and the Clostridium tetani toxin (TT). We also testedexpression of E. coli MBP, and the fragment C (TTc) and light chain(TTlight) domains of TT. MBP is not a licensed carrier, but MBPglycoconjugates has been shown to elicit polysaccharide-specific humoraland Th1-biased cellular responses in mice [19]. Similarly, the TTdomains, TTlight and TTc, have not been used in licensed vaccines, butare sufficient for protection against C. tetani challenge in mice [32].To enable glycosylation, all carriers were modified at their C-terminiwith an optimal bacterial glycosylation motif, DQNAT (SEQ ID NO: 11)[35], that was tandemly repeated four times (denoted as 4×DQNAT (SEQ IDNO: 12)). A C-terminal 6×His tag was also included to enablepurification and detection via Western blot analysis. A variant ofsuperfolder green fluorescent protein that contained an internal DQNAT(SEQ ID NO: 11) glycosylation site (sfGFP^(217-DQNAT)) was used as amodel protein to facilitate system development.

All eight carriers were synthesized in vitro with soluble yields of˜50-650 μg mL⁻¹ as determined by ¹⁴C-leucine incorporation (FIG. 2 a ).In particular, the MBP^(4×DQNAT) and PD^(4×DQNAT) variants were nearly˜100% soluble, with yields of 500 μg mL⁻¹ and 200 μg mL⁻¹, respectively,and accumulated as exclusively full-length products according to Westernblot analysis (FIG. 2 b ), making these attractive iVAX candidatesmoving forward. Notably, similar soluble yields were reached for allcarriers at 25° C., 30° C., and 37° C., with the exception ofCRM197^(4×DQNAT) (FIG. 6 a ), which is known to be heat sensitive [13].This suggests that our cell-free method for making these carriers isrobust over a 13° C.-range in biosynthesis temperature and could finduse in settings where precise temperature control is not feasible.

The open reaction environment of our cell-free reactions enabled facilemanipulation of the chemical and reaction environment to improveproduction of more complex carriers. In the case of the membrane proteinPorA^(4×DQNAT), lipid nanodiscs were added to increase solubleexpression (FIG. 7 a ). Nanodiscs provide a cellular membrane mimic toco-translationally stabilize hydrophobic regions of membrane proteins[36]. For expression of TT, which contains an intermolecular disulfidebond, expression was carried out for 2 hours in oxidizing conditions[37], which improved assembly of the heavy and light chains intofull-length product and minimized protease degradation of full-length TT(FIG. 7 b ). In vitro synthesized CRM197^(4×DQNAT) and TT^(4×DQNAT) werecomparable in size to commercially available purified diphtheria toxin(DT) and TT protein standards and were reactive with α-DT and α-TTantibodies, respectively (FIG. 8 ), indicating that both weresynthesized with immunologically relevant conformations. This is notableas CRM197 and TT are FDA-approved vaccine antigens for diphtheria andtetanus, respectively, when they are administered without conjugatedpolysaccharides. Together, our results highlight the ability of CFPS toexpress licensed conjugate vaccine carrier proteins, including thediphtheria and tetanus vaccine antigens, in soluble conformations over arange of permissible temperatures.

On-demand synthesis of bioconjugate vaccines. We next sought tosynthesize conjugated versions of these carrier proteins by mergingtheir in vitro expression with one-pot, cell-free glycosylation. For thevaccine target, we focused on the highly virulent Francisella tularensissubsp. tularensis (type A) strain Schu S4, a gram-negative, facultativecoccobacillus and the causative agent of tularemia. This bacterium iscategorized as a class A bioterrorism agent due to its high fatalityrate, low dose of infection, and ability to be aerosolized [38].Although there is currently no licensed anti-F. tularensis vaccineavailable, several studies have independently confirmed the importantrole of antibodies directed against F. tularensis LPS, specifically theO-PS repeat unit, in providing protection against the highly virulentSchu S4 strain [39-41]. More recently, a purified recombinant vaccinecomprising the F. tularensis Schu S4 O-PS conjugated to the Pseudomonasaeruginosa exotoxin A (EPA) carrier protein was produced using PGCT inliving E. coli [20, 21]. A first-generation version of this bioconjugateboosted levels of IgG specific to F. tularensis LPS and significantlyincreased the time to death upon subsequent pathogen challenge [20].Further optimization of this vaccine to enhance decoration of EPA withFtO-PS yielded a next-generation bioconjugate that was protectiveagainst challenge with the high-virulence SchuS4 strain in a ratinhalation model of tularemia [21]. In light of these earlier findings,we investigated the ability of the iVAX platform to produceanti-tularemia bioconjugate vaccine candidates on-demand via conjugationof the F. tularensis Schu S4 O-PS (FtO-PS) structure to diverse carrierproteins in vitro.

The F. tularensis Schu S4 O-PS (FtO-PS) is composed of the 826 Darepeating unit Qui4NFm-(GalNAcAN)2-QuiNAc (Qui4NFm:4,6-dideoxy-4-formamido-D-glucose; GalNAcAN:2-acetamido-2-deoxy-D-galacturonamide; QuiNAc:2-acetamido-2,6-dideoxy-D-glucose) [20, 41]. To glycosylate proteinswith FtO-PS, we produced an all-in-one iVAX lysate from glycoengineeredE. coli cells expressing the FtO-PS biosynthetic pathway and theoligosaccharyltransferase enzyme CjPg1B. This lysate, which containedlipid-linked FtO-PS and catalytically active CjP1B, was used to catalyzeiVAX reactions primed with plasmid DNA encoding sfGFP^(217-DQNAT).Control reactions in which attachment of the FtO-PS was not expectedwere performed with lysates from cells that lacked either the FtO-PSpathway or the CjPg1B enzyme. We also tested reactions lacking plasmidencoding the target protein sfGFP^(217-DQNAT) primed with plasmidencoding sfGFP^(217-DQNAT), which contained a mutated glycosylation site(AQNAT) that is not modified by CjPg1B [42]. In reactions containing theall-in-one lysate and primed with plasmid encoding sfGFP^(217-DQNAT),immunoblotting with anti-His antibody or a commercial monoclonalantibody specific to FtO-PS revealed a ladder-like banding pattern (FIG.3 a , FIG. 9 ). This ladder is characteristic of FtO-PS attachment,resulting from O-PS chain length variability through the action of theWzy polymerase [16, 20]. Glycosylation of sfGFP^(217-DQNAT) was observedonly in reactions containing a complete glycosylation pathway and thepreferred DQNAT (SEQ ID NO: 11) glycosylation sequence (FIG. 3 a ). Thisglycosylation profile was reproducible across biological replicates fromthe same lot of lysate (FIG. 3 b , left) and using different lots oflysate (FIG. 3 b , right). In vitro protein synthesis and glycosylationwas observed after 1 hour, with the amount of conjugated polysaccharidereaching a maximum between 0.75 and 1.25 hours and then decreasing,likely due to the documented instability of O antigen polysaccharidestructures upon heating [10, 12] (FIG. 6 b ). Similar glycosylationreaction kinetics are observed at 37° C., 30° C., 25° C., and roomtemperature (˜21° C.), indicating that iVAX reactions are robust over arange of temperatures (FIG. 6 c ).

Next, we asked whether FDA-approved carriers could be similarlyconjugated with FtO-PS in iVAX reactions. We also investigatedconjugation of EPA^(DNNNS-DQNRT), which has been shown to be a safe andeffective vaccine component of PGCT-derived vaccines in phase 1 clinicaltrials [43-45]. Following addition of plasmid DNA encodingMBP^(4×DQNAT), PD^(4×DQNAT), PorA^(4×DQNAT), TTc^(4×DQNAT),TTlight^(4×DQNAT), CRM197^(4×DQNAT), and EPA^(DNNNS-DQNRT),glycosylation of each with FtO-PS was observed for iVAX reactionsenriched with lipid-linked FtO-PS and CjPg1B but not control reactionslacking CjPg1B (FIG. 3 c ). We observed conjugation of high molecularweight FtO-PS species (on the order of ˜10-20 kDa) to all proteincarriers tested, which is important as glycan chain length has beenshown to play roles in the efficacy of conjugate vaccines [46, 47].Notably, our attempts to synthesize the same panel of bioconjugatesusing the established PGCT approach in living E. coli yielded lesspromising results. Specifically, we observed limited expression of thePorA membrane protein in vivo in addition to low levels of FtO-PSconjugation and reduced high molecular weight FtO-PS species inPGCT-derived conjugates compared to their iVAX-derived counterparts(FIG. 10 ). This indicates that iVAX could provide advantages over PGCTfor production of bioconjugate vaccine candidates composed of diverseand potentially membrane-bound carrier proteins with minimal requiredoptimization.

We found that reactions lasting ˜1 hour produced ˜20 μg mL⁻¹ ofglycosylated MBP^(4×DQNAT) and PD^(4×DQNAT) as determined by ¹⁴C-leucineincorporation and densitometry analysis (FIG. 11 ). At these titers, ouriVAX reactions can produce up to 20 doses per nit per hour based onrecent phase 1 clinical trials demonstrating that 1-10 μg doses ofbioconjugate vaccine candidates are well-tolerated and effective instimulating the production of antibacterial IgGs [43-45]. Hence,clinically relevant doses can be synthesized in just 1 h, making theiVAX platform an attractive option for point-of-care production orprototyping of bioconjugate vaccines.

To demonstrate the modularity of the iVAX approach for bioconjugateproduction, we sought to produce bioconjugates bearing O-PS antigensfrom ETEC E. coli strain O78 and UPEC E. coli strain O7. E. coli O78 isa major cause of diarrheal disease in developing countries, especiallyamong children, and a leading cause of traveler's diarrhea [48], whilethe O7 strain is a common cause of urinary tract infections [49]. Likethe FtO-PS, the biosynthetic pathways for EcO78-PS and EcO7-PS have beendescribed previously and confirmed to produce O-PS antigens with therepeating units GlcNAc₂Man₂ [50] and VioNAcManRhaGalGlcNAc (GlcNAc:N-acetylglucosamine; Man: mannose; VioNAc: N-acetylviosamine; Rha:rhamnose; Gal: galactose) [51], respectively. Using all-in-one iVAXlysates from cells expressing CjPg1B and either the EcO78-PS and EcO7-PSpathways in reactions primed with PD^(4×DQNAT) or sfGFP^(217-DQNAT)plasmids, we observed carrier glycosylation when both lipid-linked O-PSand CjPg1B were present in the reactions (FIG. 12 ). Collectively, ourresults demonstrate the production of bioconjugates against multiplebacterial pathogens enabled by coordinated in vitro carrier proteinsynthesis and O-PS glycosylation of licensed vaccine carrier proteins.

Portable, freeze-dried iVAX reactions containing endotoxin-editedlysates. For the iVAX technology to be an effective portable vaccineproduction platform, iVAX-derived bioconjugates must be demonstrated assafe, and iVAX reactions must be amenable to storage and distributionunder ambient conditions. A key challenge inherent in using any E.coli-based system for biopharmaceutical production is the presence oflipid A, or endotoxin, which is known to contaminate protein productsImmune recognition of lipid A by toll-like receptor 4 (TLR4) results inproduction of proinflammatory cytokines such as tumor necrosis factoralpha and interleukin-1 beta [52] that are needed to fight infection,but can cause lethal septic shock at high levels [53]. As a result, theamount of endotoxin in formulated biopharmaceuticals is regulated by theUnited States Pharmacopeia (USP), US Food and Drug Administration (FDA),and the European Medicines Agency (EMEA) [54]. Because our iVAXreactions rely on lipid-associated components, such as CjPg1B andFtO-PS, standard detoxification approaches involving the removal oflipid A [55] could compromise the activity or concentration of ourglycosylation components. Such removal strategies also increase cost andprocessing complexities, which could limit the economic accessibilityand utility of iVAX reactions in resource-limited settings.

To address this issue, we sought to modify the structure of lipid A toreduce its toxicity but maintain its adjuvanticity. For example,monophosphoryl lipid A (MPL) from Salmonella minnesotaR595 is anapproved adjuvant composed of a mixture of monophosphorylated lipids,with the primary component being pentaacylated, monophosphorylated lipidA [56]. Several groups have recently reported the ability to detoxifythe lipid A molecule through strain engineering [57, 58]. In particular,the deletion of the acyltransferase gene lpxM and the overexpression ofthe F. tularensis phosphatase LpxE in E. coli has been shown to resultin the production of nearly homogenous pentaacylated, monophosphorylatedlipid A with significantly reduced toxicity [57]. Similarly, when weproduced lysates from the CLM24 ΔlpxM strain expressing FtLpxE and theFtO-PS glycosylation pathway, we observed significantly decreased levelsof toxicity compared to wild type CLM24 lysates expressing CjPg1B andFtO-PS (FIG. 4 a ) as measured by human TLR4 activation in HEK-BluehTLR4 reporter cells [58]. It should be noted that the structuralediting of lipid A did not affect the activity of the membrane-boundCjPg1B and FtO-PS components in iVAX reactions (FIG. 13 ). Byengineering the chassis strain for lysate production, we produced iVAXlysates with endotoxin levels <1,000 EU/mL, which is within the range ofreported values for commercial protein-based vaccine products(0.288-180,000 EU/mL) [54].

A major limitation of traditional conjugate vaccines is that they mustbe refrigerated [12, 13], making it difficult to distribute thesevaccines to remote or resource-limited settings. The ability tofreeze-dry detoxified iVAX reactions for ambient temperature storage anddistribution could alleviate the logistical challenges associated withrefrigerated supply chains that are required for existing vaccines. Toinvestigate this possibility, identical iVAX reactions producingsfGFP^(217-DQNAT) were run immediately or following lyophilization andrehydration. In both cases, FtO-PS is attached to the target proteinwhen CjPg1B was present in the reaction (FIG. 4 b ). Detoxified,freeze-dried iVAX reactions can be scaled to 5 mL for reproducibleFtO-PS-conjugated MBP^(4×DQNAT) and PD^(4×DQNAT) with modificationefficacies similar to those observed without freeze-drying (FIG. 14 ).The ability to lyophilize iVAX reactions without compromisingbioconjugate synthesis levels highlights the potential for portable,on-demand vaccine production.

In vitro synthesized bioconjugates elicit bactericidal pathogen-specificantibodies in mice. To validate the efficacy of bioconjugates producedusing the iVAX platform, we next evaluated the ability of iVAX-derivedbioconjugates to elicit anti-FtLPS antibodies in mice. Importantly, wefound that BALB/c mice receiving iVAX-derived FtO-PS-conjugatedMBP^(4×DQNAT) and PD^(4×DQNAT) produced high titers of FtLPS-specificIgG antibodies, which were significantly elevated compared to the titersmeasured in the sera of control mice receiving PBS or aglycosylatedMBP^(4×DQNAT) and PD^(4×DQNAT) carrier proteins (FIG. 5 a , FIG. 15 ).Interestingly, the IgG titers measured in sera from mice receivingglycosylated MBP^(4×DQNAT) derived from PGCT were similar to the titersobserved in the control groups (FIG. 5 a , FIG. 15 ), in line with theweaker glycosylation of this candidate relative to its iVAX-derivedcounterpart (FIG. 10 ). Notably, both MBP and PD bioconjugates producedusing iVAX elicited similar levels of IgG production and neitherresulted in any observable adverse events in mice, confirming themodularity and safety of the technology for producing bioconjugatevaccine candidates.

We further characterized IgG titers by analysis of IgG1 and IgG2asubtypes and found that iVAX-derived FtO-PS-conjugated MBP^(4×DQNAT) andPD^(4×DQNAT) boosted production of IgG1 antibodies by >2 orders ofmagnitude relative to all control groups and to glycosylatedMBP^(4×DQNAT) derived from PGCT (FIG. 5 b ). This analysis also revealedthat our iVAX-derived bioconjugates elicited a strongly Th2-biased(IgG1>>IgG2a) response, which is characteristic of most conjugatevaccines [59]. Taken together, these results provide clear evidence thatthe iVAX platform supplies vaccine candidates that are capable ofeliciting strong, pathogen-specific humoral immune responses andrecapitulate the Th2 bias that is characteristic of licensed conjugatevaccines.

To determine if the IgG antibodies elicited by the iVAX-derivedbioconjugates were functional, we next screened mouse sera for F.tularensis killing activity using a serum bactericidal assay (SBA). Miceimmunized with iVAX-derived bioconjugates clearly had opsonically activeantibodies that mediated killing of F. tularensis LVS Iowa, whereas micereceiving empty OMVs or PBS had significantly less killing activity.

Discussion

In this work we have established iVAX, a cell-free platform forportable, on-demand production of bioconjugate vaccines. We show thatiVAX reactions can be detoxified to ensure the safety of bioconjugatevaccine products, freeze-dried for cold chain-independent distribution,and re-activated by simply adding water. As a model vaccine candidate,we show that anti-F. tularensis bioconjugates derived from freeze-dried,endotoxin-edited iVAX reactions elicited pathogen-specific IgGantibodies in mice as part of a Th2-biased immune responsecharacteristic of licensed conjugate vaccines.

The iVAX platform has several exciting features. First, iVAX is modular,which we have demonstrated through the interchangeability of (i) carrierproteins, including those used in licensed conjugate vaccines, and (ii)bacterial O-PS antigens from F. tularensis subsp. tularensis (type A)Schu S4, ETEC E. coli O78, and UPEC E. coli O7. Further expansion of theO-PS pathways used in iVAX should be relatively straightforward giventhe commonly observed clustering of polysaccharide biosynthetic genes inthe genomes of pathogenic bacteria [60]. This feature should make iVAXan attractive option for rapid, de novo development of bioconjugatevaccine candidates in response to a disease outbreak or against emergingdrug resistant bacteria.

Second, iVAX reactions are inexpensive, costing ˜$11.75 mL⁻¹ (data notshown) with the ability to synthesize ˜20 μg bioconjugate mL⁻¹ (FIG. 11). Assuming a dose size of 10 μg, our iVAX reactions can produce avaccine dose for S5.88. For comparison, the CDC cost per dose forconjugate vaccines ranges from $9.23 for the TT conjugate ActHIB® to$73.83 and $131.77 for the CRM197 conjugates Menveo® and Prevnar 13®[61]. We anticipate that the low cost of the iVAX platform willencourage its adoption.

Third, and rather interestingly, we observed that iVAX-derivedbioconjugates were significantly more effective at elicitingFtLPS-specific IgGs than a bioconjugate derived from living E. colicells using PGCT (FIG. 5 ). One possible explanation for this increasedeffectiveness is the more extensive glycosylation that we observed forthe in vitro expressed carriers, with greater carbohydrate loading perprotein and decoration with a broader range of higher molecular weightFtO-PS species compared to their PGCT-derived counterparts. This isconsistent with previous reports of PGCT-derived anti-F. tularensisbioconjugates which show that increasing the ratio of glycan to proteinin bioconjugates result in enhanced protection against F. tularensis ina rat inhalation model of tularemia [21]. A number of studies havedemonstrated that the degree of epitope density and glycan chain lengthare critical factors influencing the magnitude of the ensuingepitope-specific immune response [46, 47, 62-64]. These results as wellas our own point to the fact that a deeper understanding of importantimmunogen design features, which could be readily controlled and variedusing the iVAX platform, will be crucial to producing better defined,more effective conjugate vaccine candidates in the future.

In summary, iVAX provides an exciting new approach for portable,on-demand biomanufacturing of conjugate vaccines. A key feature of iVAXis that seamlessly integrates CFPS and glycosylation with bacterialpolysaccharide antigens in one-pot reactions that can be freeze-driedfor refrigeration-free storage and transportation, and can bere-activated for point-of-use vaccine synthesis simply by adding water.This not only alleviates cold chain requirements, which is important fordelivering medicines on demand in regions with limited infrastructureand could minimize vaccine losses due to spoilage, but also provides aunique means for rapidly responding to pathogen outbreaks and emergentthreats. As a result, we believe that the iVAX technology platform,along with an emerging set of collective efforts in making biomedicineson-demand [24, 25, 65-67], have the potential for promoting betteraccess to costly drugs through decentralized production.

Methods

Bacterial strains and plasmids. NEB 5-alpha was used for plasmid cloningand purification. The CLM24 or CLM24 ΔlpxM strains were used as thesource strain for preparing lysates with and without selectivelyenriched glycosylation components. CLM24 was used as the cassis forexpressing bioconjugates in vivo using PGCT. CLM24 is a glyco-optimizedderivative of W3110 that carries a deletion in the gene encoding theWaaL ligase, thus facilitating the accumulation of preassembled glycanson Und-PP [16]. CLM24 ΔlpxM has an endogenous acyltransferase deletionand serves as the chassis strain for production of detoxified lysates.

The CLM24 ΔlpxM strain was generated using the Datsenko-Wanner geneknockout method [68]. Briefly, CLM24 cells were transformed with thepKD46 plasmid encoding the λ red system. Transformants were grown to anOD₆₀₀ of 0.5-0.7 in 25 mL LB-Lennox media (10 g L⁻¹ tryptone, 5 g L⁻¹yeast extract and 5 g L⁻¹ NaCl) with 50 μg mL⁻¹ carbenicillin at 30° C.harvested and washed three times with 25 mL ice-cold 10% glycerol tomake them electrocompetent, and resuspended in a final volume of 100 μL10% glycerol. In parallel, a lpxM knockout cassette was generated by PCRamplifying the kanamycin resistance cassette from pKD4 with forward andreverse primers with homology to lpxM. Electrocompetent cells weretransformed with 400 ng of the lpxM knockout cassette and plated on LBagar with 30 μg mL⁻¹ kanamycin for selection of resistant colonies.Plates were grown at 37° C. to cure cells of the pKD46 plasmid. Coloniesthat grew on kanamycin were confirmed to have acquired the knockoutcassette via colony PCR and DNA sequencing. These confirmed colonieswere then transformed with pCP20 to remove the kanamycin resistance genevia Flp-FRT recombination. Transformants were plated on LB agar with 50μg mL⁻¹ carbenicillin. Following selection, colonies were grown inliquid culture at 42° C. to cure cells of the pCP20 plasmid. Colonieswere confirmed to have lost both lpxM and the knockout cassette viacolony PCR and DNA sequencing and confirmed to have lost both kanamycinand carbenicillin resistance via replica plating on LB agar plates withcarbenicillin and kanamycin. To enable overexpression of F. tualrensisLpxE, the lpxE gene was inserted into the pSF CjPg1B vector downstreamof pglB via Gibson assembly. Lysate detoxification resulting fromdeletion of lpxM and overexpression of LpxE was confirmed using the TLR4activation assay described below.

Plasmids pJL1-MBP^(4×DQNAT), pJL1-PD^(4×DQNAT), pJL1-PorA^(4×DQNAT),pJL1-TTc^(4×DQNAT), pJL1-TTlight^(4×DQNAT), pJL1-CRM197^(4×DQNAT),pJL1-TT^(4×DQNAT) and generated via PCR amplification and subsequentGibson Assembly of a codon optimized gene construct purchased from IDTwith a C-terminal 4×DQNAT (SEQ ID NO: 12)-6×His tag between the NdeI andSalI restriction sites in the pJL1 vector. PlasmidpJL1-EPA^(DNNNS-DQNRT) was constructed using the same approach, butwithout the addition of a C-terminal 4×DQNAT (SEQ ID NO: 12)-6×His tag.Plasmids pTrc99s-ssDsbA-MBP^(4×DQNAT), pTrc99s-ssDsbA-PD^(4×DQNAT),pTrc99s-ssDsbA-PorA^(4×DQNAT), pTrc99s-ssDsbATTc^(4×DQNAT),pTrc99s-ssDsbA-TTlight^(4×DQNAT), and pTrc99s-ssDsbA-EPA^(4×DQNAT) werethen created via PCR amplification of each carrier protein gene andinsertion into the pTrc99s vector between the Ncol and HindIIIrestriction sites via Gibson Assembly. Plasmid pSF-CjPg1B-LpxE wasconstructed using a similar approach, but via insertion of the lpxE genefrom pE [58] between the NdeI and NsiI restriction sites in the pSFvector. Inserts were amplified via PCR using Phusion® High-Fidelity DNApolymerase (NEB) with forward and reverse primers designed with theNEBuilder® Assembly Tool (nebuilder.neb.com) and purchased from IDT. ThepJL1 vector (Addgene 69496) was digested using restriction enzymes NdeIand SalI-HF® (NEB). The pSF vector was digested using restrictionenzymes NdeI and NotI (NEB). PCR products were gel extracted using anEZNA Gel Extraction Kit (Omega Bio-Tek), mixed with Gibson assemblyreagents and incubated at 50° C. for 1 hour. Plasmid DNA from the Gibsonassembly reactions were transformed into E. coli NEB 5alpha cells andcircularized constructs were selected using kanamycin at 50 μg ml⁻¹(Sigma). Sequence-verified clones were purified using an EZNA PlasmidMidi Kit (Omega Bio-Tek) for use in FD-CF reactions.

S30 lysate preparation. E. coli CLM24 source strains were grown in 2×YTPmedia (10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl, 7 g/L K₂HPO₄,3 g/L KH₂PO₄, pH 7.2) in shake flasks (1 L scale) or a Sartorius StedimBIOSTAT Cplus bioreactor (10 L scale) at 37° C. Protein synthesis yieldsand glycosylation activity were reproducible across different batches oflysate at both small (FIG. 3 b ) and large scale (FIG. 14 a ). Togenerate CjPg1B-enriched lysate, CLM24 cells carrying plasmid pSF-CjPg1B[69] was used as the source strain. To generate FtO-PS-enriched lysates,CLM24 carrying plasmid pGAB2 [20] was used as the source strain. Togenerate one-pot lysates containing both CjPg1B and the FtO-PS,EcO78-PS, or EcO7-PS, CLM24 carrying pSF-CjPg1B and one of the followingbacterial O-PS biosynthetic pathways was used as the source strain:pGAB2 (FtO-PS), pMW07-O78 (EcO78-PS), and pJHCV32 (EcO7-PS). CjPg1Bexpression was induced at an OD₆₀₀ of 0.8-1.0 with 0.02% (w/v)L-arabinose and cultures were moved to 30° C. Cells were grown to afinal OD₆₀₀ of 3.0, at which point cells were pelleted by centrifugationat 5,000×g for 15 mM at 4° C. Cell pellets were then washed three timeswith cold S30 buffer (10 mM Tris-acetate pH 8.2, 14 mM magnesiumacetate, 60 mM potassium acetate) and pelleted at 5000×g for 10 mM at 4°C. After the final wash, cells were pelleted at 7000×g for 10 mM at 4°C., weighed, flash frozen in liquid nitrogen, and stored at −80° C. Tomake cell lysate, cell pellets were resuspended to homogeneity in 1 mLof S30 buffer per 1 g of wet cell mass. Cells were disrupted via asingle passage through an Avestin EmulsiFlex-B15 (1 L scale) orEmulsiFlex-C3 (10 L scale) high-pressure homogenizer at 20,000-25,000psi. The lysate was then centrifuged twice at 30,000×g for 30 mM toremove cell debris. Supernatant was transferred to clean microcentrifugetubes and incubated at 37° C. with shaking at 250 rpm for 60 minFollowing centrifugation (15,000×g) for 15 mM at 4° C., supernatant wascollected, aliquoted, flash-frozen in liquid nitrogen, and stored at−80° C. S30 lysate was active for about 3 freeze-thaw cycles andcontained ˜40 g/L total protein as measured by Bradford assay.

Cell-free protein synthesis. CFPS reactions were carried out in 1.5 mLmicrocentrifuge tubes (15 μL scale), 15 mL conical tubes (1 mL scale),or 50 mL conical tubes (5 mL scale) with a modified PANOx-SP system[70]. The CFPS reaction mixture consists of the following components:1.2 mM ATP; 0.85 mM each of GTP, UTP, and CTP; 34.0 μg mL⁻¹L-5-formyl-5, 6, 7, 8-tetrahydrofolic acid (folinic acid); 170.0 mgmL⁻⁻¹ of E. coli tRNA mixture; 130 mM potassium glutamate; 10 mMammonium glutamate; 12 mM magnesium glutamate; 2 mM each of 2.0 aminoacids; 0.4 mM nicotinamide adenine dinucleotide (NAD); 0.27 mMcoenzyme-A (CoA); 1.5 mM spermidine; 1 mM putrescine; 4 mM sodiumoxalate; 33 mM phosphoenolpyruvate (PEP); 57 mM HEPES; 13.3 μg mLplasmid; and 27% v/v of cell lysate. For reaction volumes mL, plasmidwas added at 6.67 μg mL⁻¹, as this lower plasmid concentration conservedreagents with no effect on protein synthesis yields or kinetics (FIG. 16). For expression of PorA, reactions were supplemented with Nanodiscs at1 μg mL⁻¹, which were prepared as previously described [33] or purchased(Cube Biotech). For expression of CRM197^(4×DQNAT), CFPS was carried outat 25° C. for 20 hours, unless otherwise noted. For all other carrierproteins, CFPS was run at 30° C. for 20 hours, unless otherwise noted.

For expression of TT^(4×DQNAT), which contains intermolecular disulfidebonds, CFPS was carried out under oxidizing conditions. For oxidizingconditions, lysate was pre-conditioned with 750 μM iodoacetamide at roomtemperature for 30 min to covalently bind free sulfhydryls (—SH) and thereaction mix was supplemented with 200 mM glutathione at a 4:1 ratio ofoxidized and reduced forms and 60.5 μM recombinant E. coli DsbC [37].

In vitro bioconjugate vaccine expression (iVAX). For in vitro expressionand glycosylation of acceptor proteins in crude lysates, a two-phasescheme was implemented. In the first phase, CFPS was carried out for 15mM at 25-30° C. as described above. In the second phase, proteinglycosylation was initiated by the addition of MnCl₂ and DDM at a finalconcentration of 25 mM and 0.1% (w/v), respectively, and allowed toproceed at 30° C. for a total reaction time of 1-2 hours. Proteinsynthesis yields and glycosylation activity were reproducible acrossbiological replicates of iVAX reactions at both small (FIG. 3 b ) andlarge scale (FIG. 14 b ). Reactions were then centrifuged at 20,000×gfor 10 min to remove insoluble or aggregated protein products and thesupernatant was analyzed by SDS-PAGE and Western blotting.

Purification of aglycosylated and glycosylated carriers from iVAXreactions was carried out using Ni-NTA agarose (Qiagen) according tomanufacturer's protocols. Briefly, 0.5 mL Ni-NTA agarose per 1 mLcell-free reaction mixture was equilibrated in Buffer 1 (300 mM NaCl 50mM NaH₂PO₄) with 10 mM imidazole. Soluble fractions from iVAX reactionswere loaded on Ni-NTA agarose and incubated at 4° C. for 2-4 hours tobind 6×His-tagged protein. Following incubation, the cell-freereaction/agarose mixture was loaded onto a polypropylene column (BioRad)and washed twice with 6 column volumes of Buffer 1 with 20 mM imidazole.Protein was eluted in 4 fractions, each with 0.3 mL Buffer 1 with 300 mMimidazole per mL of cell-free reaction mixture. All buffers were usedand stored at 4° C. Protein was stored at a final concentration of 1-2mg/mL in sterile 1×PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mMKH₂PO₄, pH 7.4) at 4° C.

Expression of bioconjugates in vivo using protein glycan couplingtechnology (PGCT). Plasmids encoding glycoconjugate carrier genepreceded by the DsbA leader sequence for translocation to the periplasmwere transformed into CLM24 cells carrying pGAB2 and pSF-CjPg1B. CLM24carrying only pGAB2 was used as a negative control. Transformed cellswere grown in 5 mL LB media (10 g L⁻¹ yeast extract, 5 g L⁻¹ tryptone, 5g L⁻¹ NaCl) overnight at 37° C. The next day, cells were subculturedinto 100 mL LB and allowed to grow at 37° C. for 6 hours after which theculture was supplemented with 0.2% arabinose and 0.5 mM IPTG to induceexpression of CjPg1B and the bioconjugate carrier protein, respectively.Protein expression was then carried out for 16 h at 30° C., at whichpoint cells were harvested. Cell pellets were resuspended in 1 mLsterile 1×PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH7.4) and lysed using a Q125 Sonicator (Qsonica, Newtown, Conn.) at 40%amplitude in cycles of 10 s on/10 s off for a total of 5 mM Solublefractions were isolated following centrifugation at 15,000 rpm for 30 mMat 4° C. Protein was purified from soluble fraction using Ni-NTA spincolumn (Qiagen) following manufacturer's protocol.

Quantification of cell-free protein synthesis yields. To quantify theamount of protein synthesized in iVAX reactions, two approaches wereused. Fluorescence units of sfGFP were converted to concentrations usinga previously reported standard curve [71]. Yields of all other proteinswere assessed via the addition of 10 μM L-¹⁴C-leucine (11.1 GBq mmol⁻¹,PerkinElmer) to the CFPS mixture to yield trichloroaceticacid-precipitable radioactivity that was measured using a liquidscintillation counter as described previously [72]. These reactions werealso run on a Coomassie-stained SDS-PAGE gel and exposed byautoradiography. Autoradiographs were imaged with a Typhoon 7000 (GEHealthcare Life Sciences).

Western blot analysis. Samples were run on 4-12% Bis-Tris SDS-PAGE gels(Invitrogen). Following electrophoretic separation, proteins weretransferred from gels onto Immobilon-P polyvinylidene difluoride (PVDF)membranes (0.45 μm) according to the manufacturer's protocol. Membraneswere washed with PBS (80 g L⁻¹ NaCl, 0.2 g L⁻¹ KCl, 1.44 g L⁻¹ Na₂HPO₄,0.24 g L⁻¹ KH₂PO₄, pH 7.4) followed by incubation for 1 h in Odyssey®Blocking Buffer (LiCor). After blocking, membranes were washed 6 timeswith PBST (80 g L⁻¹ NaCl, 0.2 g L⁻¹ KCl, 1.44 g L⁻¹ Na₂HPO₄, 0.24 g L⁻¹KH₂PO₄, 1 mL L⁻¹ Tween-20, pH 7.4) with a 5 mM incubation between eachwash. For iVAX samples, membranes were probed with both an anti-6×Histag antibody and an anti-O-PS antibody or antisera specific to the Oantigen of interest, if commercially available. Probing of membranes wasperformed for at least 1 hour with shaking at room temperature, afterwhich membranes were washed with PBST in the same manner as describedabove and probed with fluorescently labeled secondary antibodies.Membranes were imaged using an Odyssey® Fc imaging system (LiCor).CRM197 and TT production were compared to commercial DT and TT standards(Sigma) and orthogonally detected by an identical SDS-PAGE procedurefollowed by Western blot analysis with a polyclonal antibody thatrecognizes diphtheria or tetanus toxin, respectively.

TLR4 activation assay. HEK-Blue hTLR4 cell lines were purchased fromInvivogen and maintained according to the manufacturer's specifications.Cells were maintained in DMEM media, high glucose/L-glutamine supplementwith 10% FBS, 50 U mL⁻¹ penicillin, 50 mg mL⁻¹ streptomycin, and 100 μgNormacin™ at 37° C. in a humidified atmosphere containing 5% CO₂. Afterreaching ˜50-80% confluency, cells were plated into 96-well plates at adensity of 1.4×10⁵ cells per mL in HEK-Blue detection media (Invivogen).Antigens were added at the following concentrations: 100 ng μL⁻¹purified protein; and 100 ng μL⁻¹ total protein in lysate. Purified E.coli O55:B5 LPS (Sigma-Aldrich) and detoxified E. coli O55:B5(Sigma-Aldrich) were added at 1.0 ng mL⁻¹ and served as positive andnegative controls, respectively. Plates were incubated at 37° C., 5% CO₂for 10-16 h, before being analyzed using a microplate reader at 620 nm.Statistical significance was determined using paired t-tests.

Mouse immunization. Nine groups of six-week old BALB/c mice (HarlanSprague Dawley) were injected subcutaneously with 100 μL PBS (pH 7.4)alone or containing purified aglycosylated MBP, FtO-PS-conjugated MBP,aglycosylated PD, or FtO-PS-conjugated PD, as previously described [73].Groups were composed of six mice except for the PBS control group, whichwas composed of five mice. The amount of antigen in each preparation wasnormalized to 7.5 μg to ensure that an equivalent amount ofaglycosylated protein or bioconjugate was administered in each case.Purified protein groups formulated in PBS were mixed with an equalvolume of incomplete Freund's Adjuvant (Sigma-Aldrich) before injection.Prior to immunization, material for each group (5 μL) was streaked on LBagar plates and grown overnight at 37° C. to confirm sterility andendotoxin activity was measured by TLR4 activation assay. Each group ofmice was boosted with an identical dosage of antigen 21 d and 42 d afterthe initial immunization. Blood was obtained on d −1, 21, 35, 49, 63 viasubmandibular collection and at study termination on d 70 via cardiacpuncture. Mice were observed 24 and 48 h after each injection forchanges in behavior and physical health and no abnormal responses wereobserved. This study and all procedures were done in accordance withProtocol 2012-0132 approved by the Cornell University InstitutionalAnimal Care and Use Committee.

Enzyme-linked immunosorbent assay. F. tularensis LPS-specific antibodiesproduced in immunized mice were measured via indirect ELISA using amodification of a previously described protocol [73]. Briefly, sera wereisolated from the collected blood draws after centrifugation at 5,000×gfor 10 min and stored at −20° C.; 96-well plates (Maxisorp; NuncNalgene) were coated with F. tularensis LPS (BEI resources) at aconcentration of 5 μg mL⁻¹ in PBS and incubated overnight at 4° C. Thenext day, plates were washed three times with PBST (PBS, 0.05% Tween-20,0.3% BSA) and blocked overnight at 4° C. with 5% nonfat dry milk(Carnation) in PBS. Samples were serially diluted by a factor of two intriplicate between 1:100 and 1:12,800,000 in blocking buffer and addedto the plate for 2 h at 37° C. Plates were washed three times with PBSTand incubated for 1 h at 37° C. in the presence of one of the followingHRP-conjugated antibodies (all from Abcam and used at 1:25,000dilution): goat anti-mouse IgG, anti-mouse IgG1, and anti-mouse IgG2a.After three additional washes with PBST, 3,3′-5,5′-tetramethylbenzidinesubstrate (1-Step Ultra TMB-ELISA; Thermo-Fisher) was added, and theplate was incubated at room temperature in the dark for 30 min. Thereaction was halted with 2 M H₂SO₄, and absorbance was quantified viamicroplate spectrophotometer (Tecan) at a wavelength of 450 nm. Serumantibody titers were determined by measuring the lowest dilution thatresulted in signal 3 SDs above no serum background controls. Statisticalsignificance was determined in RStudio 1.1.463 using one-way ANOVA andthe Tukey-Kramer post hoc honest significant difference test.

Serum bactericidal assay. SBA was performed using a modified version ofpreviously reported protocols [74, 75]. An overnight culture of F.tularensis LVS Iowa was used to make a suspension of 10⁵ cfu/mL insterile PBS. Serum samples taken 10 weeks after immunization fromunchallenged mice were heat inactivated via incubation at 56° C. for 30min before use. Human mAb F598 (1 mg/mL=1:1 dilution of mAb) was used asa positive control for bactericidal activity. Serum samples and mAb F598were diluted 1:2 in sterile PBS and then serially diluted threefold to amaximum dilution factor of 1:54 before being diluted 1:5 in the assay(final concentration range 1:10-1:270). Naïve baby rabbit complement(Cedarlane) or heat-inactivated complement (56° C. for 30 min) was usedat a final concentration of 6% (vol/vol). Reactions were incubated at37° C. for 30 min before 10 μL of each reaction was spotted on cysteineheart agar+9% sheep's blood and allowed to trail down one-half thelength of the plate to facilitate colony counting. Plates were thenincubated overnight at 37° C., and colonies were counted the next day.Percentage of bactericidal activity was calculated by comparing thenumber of cfu in a spot with the number of cfu in the spot of the “nocomplement” control.

Statistical analysis. Statistical parameters including the definitionsand values of n, standard deviations, and standard errors are reportedin the Figures and corresponding Figure Legends. Analytical techniquesare described in the corresponding Methods section.

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PATENT REFERENCES

U.S. Pat. Nos. 4,496,538; 4,727,136; 5,478,730; 5,556,769; 5,623,057;5,665,563; 5,679,352; 6,168,931; 6,248,334; 6,518,058; 6,783,957;6,869,774; 6,994,986; 7,118,883; 7,189,528; 7,338,789; 7,387,884;7,399,610; 8,703,471; and 8,999,668; U.S. Publication Nos. 2005/0170452;2006/0211085; 2006/0234345; 2006/0252672; 2006/0257399; 2006/0286637;2007/0026485; 2007/0178551; 2015/0259757; 2016/0060301; 2016/0362708;2017/0349928; 20180016614; 2018/0044905; International Publication Nos.WO2003/056914A1; WO2004/013151A2; WO2004/035605A2; WO2006/102652A2;WO2006/119987A2; and WO2007/120932A2; the contents of which areincorporated herein by reference in their entireties.

In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. A method for synthesizing an N-glycosylated carrier proteinfor a polysaccharide via coordinated transcription, translation, andN-glycosylation in vitro, in a single vessel, the method comprising:transcribing and translating a carrier protein in a cell-free proteinsynthesis reaction, the carrier protein comprising an inserted consensussequence, D/E-X₁-N-X₂-SIT or N-X-S/T, wherein X, X₁ and X₂ may be anynatural or unnatural amino acid except proline; (ii) glycosylating thecarrier protein in the cell-free protein synthesis reaction with atleast one polysaccharide, wherein the at least one polysaccharide is atleast one bacterial O-antigen; wherein the single vessel comprises atemplate encoding the carrier protein, an exogenous lipid-linkedoligosaccharide (LLO) comprising the bacterial O-antigen, and one ormore Escherichia coli (E. coli) cell lysates from engineered E. colistrains, wherein the engineered E. coli strains comprise: (a) a nucleicacid encoding an orthogonal or heterologous oligosaccharyltransferase(OST) which is expressed in the engineered E. coli strains; (b) one orboth of: (1) a mutation of the endogenous lpxM gene wherein the mutationresults in the reduced expression or and/or activity of the encodedmyristoyltransferase; (2) a nucleic acid comprising an orthogonal orheterologous LpxE gene and encoding a lipidA 1-phosphatase which isexpressed in the engineered E. coli strains; (c) a mutation in theendogenous waaL gene, wherein the mutation results in the reducedexpression and/or activity of the encoded O-antigen ligase; (d)prokaryotic transcription and translation machinery.
 2. The method ofclaim 1, wherein the bacterial O-antigen is from E. coli.
 3. The methodof claim 1, wherein the bacterial O-antigen is from Franciscellatularensis.
 4. The method of claim 1, further comprising formulating theN-glycosylated carrier protein as an antigenic composition comprisingthe N-glycosylated carrier protein.
 5. The method of claim 1, furthercomprising formulating the N-glycosylated carrier protein as a vaccinecomposition comprising the N-glycosylated carrier protein.
 6. The methodof claim 5, wherein the vaccine composition further comprises anadjuvant.
 7. The method of claim 1, wherein the carrier protein is anengineered variant of E. coli maltose binding protein (MBP).
 8. Themethod of claim 1, wherein the carrier protein is selected from adetoxified variant of the toxin from Clostridium tetani, a detoxifiedvariant of the toxin from Corynebacterium diptheriae, Haemophilusinfluenzae protein D (PD) or a variant thereof, and Neisseriameningitidis porin protein (PorA) or a variant thereof.
 9. The method ofclaim 1, wherein the glycosylating step utilizes anoligosaccharyltransferase (OST) which is a naturally occurring bacterialhomolog of C. jejuni Pg1B, and wherein the consensus sequence comprisesD/E-X₁-N-X₂-S/T.
 10. The method of claim 1, wherein the glycosylatingstep utilizes an OST that is an engineered variant of C. jejuni Pg1B,and wherein the consensus sequence comprises D/E-X₁-N-X₂-S/T.
 11. Themethod of claim 1, wherein the wherein the glycosylating step utilizesan OST that is a naturally occurring archaeal OST, and wherein theconsensus sequence comprises N-X-S/T.
 12. The method of claim 1, whereinthe wherein the glycosylating step utilizes an OST which is a naturallyoccurring single-subunit eukaryotic OST, and wherein the consensussequence comprises N-X-S/T.
 13. The method of claim 1, wherein theheterologous LpxE gene is from F. tularensis.
 14. The method of claim 1,wherein the one or more cell lysates have an endotoxin unit (EU)concentration of less than about 180,000 EU/ml.
 15. A method ofvaccinating a subject in need thereof against a bacterial O-antigen, themethod comprising administering to the subject the vaccine compositionof claim 5.